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Inhibition kinetics on carboxylesterase and acetylcholinesterase of Liposcelis bostrychophila and Liposcelis entomophila (Psocop., Liposcelididae) of two insecticides
Abstract
The inhibition kinetics on carboxylesterase (CarE) and acetylcholinesterase (AChE) of Liposcelis bostrychophila and Liposcelis entomophila of paraoxon and carbosulfan were compared. The results showed that L. entomophila exhibited significantly greater specific activity of CarE than L. bostrychophila [0.045 vs. 0.012 μmol of α-naphthyl acetate (α-NA) hydrolysed/mg protein/min]. Moreover, L. entomophila showed higher affinity (i.e. lower Km value) to the substrate α-NA than L. bostrychophila (0.29 vs. 0.67 mm). For AChE-specific activity and affinity, no significant differences between the two species were observed. Based on the I50 values, AChE of L. bostrychophila was more sensitive to paraoxon and carbosulfan than that of L. entomophila. According to inhibition kinetics, the results revealed that AChE of L. bostrychophila was 3.8-fold more sensitive to inhibition by paraoxon than that of L. entomophila, but L. entomophila was 1.5-fold more susceptible to carbosulfan.
... It works on nicotinic acetyl choline receptors (Liu et al., 2005) and hyper stimulate the nervous tissue which leads to the death of insect (Bloomquist, 2009). According to Cheng et al., (2004) ...
... According to Cheng et al., (2004) ALP activity has been inhibited 32.8% when mealworm pupae indirectly treated with Deodar oil. ...
This study was conducted to evaluate the biochemical changes in the pupae of mealworm treated with LC50 concentration of Deodar oil in comparison to imidacloprid and carbosulfan insecticides. The larvae of mealworm beetle were treated with deodar oil (3.41 %), carbosulfan (0.086 %) and imidacloprid (0.023 %) through feeding method. The trerated larvae when able to reach the pupal stage successfully were crushed at the pupal stage for the biochemical analysis. Deodar oil and carbosulfan inhibited cholinesterase activity while imidacloprid enhanced this activity in the pupae of mealworm. ALP activity was inhibited with Deodar oil while enhanced when treated with imidacloprid and carbosulfan in the pupae of mealworm. It is concluded that the chronic effects of insecticides are carried out in the next developmental stage of mealworm pest from larvae to pupae. Deodar oil, a biopesticide has a strong biochemical effect on pupae of mealworm pest by altering their enzymatic activities. Deodar oil is environment friendly and can be effectively used as a substitute of synthetic insecticides.
... It works on nicotinic acetyl choline receptors (Liu et al., 2005) and hyper stimulate the nervous tissue which leads to the death of insect (Bloomquist, 2009). According to Cheng et al., (2004) ...
... According to Cheng et al., (2004) ALP activity has been inhibited 32.8% when mealworm pupae indirectly treated with Deodar oil. ...
The aim of the present study was to evaluate the toxicological potential of the methanol extract of Elaeagnus angustifolia plant against the 3 rd instar larvae of Drosophila melanogaster megin (Diptera/Drosophilidae) by contact method. The larvae were exposed to the treatment of the studied extract for 24, 48, 72, 96 and 120 hours intervals. The mortality rate of larvae, retrieval of pupae and emergence of adults were noted at each interval of treatment. The obtained results indicated that methanol extract of E. angustifolia possess toxic potential against D. melanogaster larvae. With 1, 2 and 3% concentration of the studied extract, mortality rate was found to be 10, 10, 10 (for 24 hours), 10, 30, 90 (for 48 hours), 10, 50, 90 (for 72 hours), 10, 60, 90 (for 96 hours) and 10, 60, 90 (for 120 hours) respectively. The mortality rate of the larvae indicated that toxicity of the extract increases linearly with the increase of concentration and time period of treatment. This extract can be used against dipterous larvae and for the control of insect pests. The use of botanical extracts as a tool for biological control of pests is environment friendly and has no impaired health risks unlike those associated with chemical control measures. In this way, it is concluded that extract of E. angustifolia has toxic potential and can be effectively used for the control of insect pests.
... It works on nicotinic acetyl choline receptors (Liu et al., 2005) and hyper stimulate the nervous tissue which leads to the death of insect (Bloomquist, 2009). According to Cheng et al., (2004) ...
... According to Cheng et al., (2004) ALP activity has been inhibited 32.8% when mealworm pupae indirectly treated with Deodar oil. ...
Abstract
This study was conducted to evaluate the biochemical changes in the pupae of mealworm treated with LC50
concentration of Deodar oil in comparison to imidacloprid and carbosulfan insecticides. The larvae of mealworm
beetle were treated with deodar oil (3.41 %), carbosulfan (0.086 %) and imidacloprid (0.023 %) through feeding
method. The trerated larvae when able to reach the pupal stage successfully were crushed at the pupal stage for the
biochemical analysis. Deodar oil and carbosulfan inhibited cholinesterase activity while imidacloprid enhanced this
activity in the pupae of mealworm. ALP activity was inhibited with Deodar oil while enhanced when treated with
imidacloprid and carbosulfan in the pupae of mealworm. It is concluded that the chronic effects of insecticides are
carried out in the next developmental stage of mealworm pest from larvae to pupae. Deodar oil, a biopesticide has a
strong biochemical effect on pupae of mealworm pest by altering their enzymatic activities. Deodar oil is
environment friendly and can be effectively used as a substitute of synthetic insecticides.
... AChE exists in the presynaptic membrane, postsynaptic membrane, and synaptic cleft and can hydrolyze acetylcholine to maintain the normal transmission of nerve impulses [50]. AChE activity is inhibited by xenobiotics, including Triadica sebifera, Basilicum ocimum, and Origanum marjorana [51,52], which results in the accumulation of acetylcholine in the insect body, triggering excitotoxicity [53]. In this study, acetamiprid inhibited aphid CarE activity, which is consistent with previous reports [54]. ...
Simple Summary
Aphis craccivora is an important pest affecting various crops worldwide. Lecanicillium spp. are important entomopathogenic fungi of Hypocreales. However, there have been few studies on the infection of A. craccivora by Lecanicillium and the related insecticidal mechanisms. In this study, Lecanicillium araneicola HK-1 was shown to produce protease and chitinase to degrade the integument of A. craccivora, resulting in insect’s death because of the synergistic effects of parasitism and harmful metabolites of the strain HK-1.
Abstract
Aphis craccivora (Hemiptera: Aphididae) is an important pest affecting various crops worldwide. However, only few studies have been conducted on the infection of A. craccivora by Lecanicillium and related insecticidal mechanisms. We investigated the infection process of A. craccivora by Lecanicillium araneicola HK-1 using fluorescence microscopy and scanning electron microscopy (SEM), and our results indicated that the conidia of strain HK-1 easily attached to the feet and dorsum of A. craccivora. The activities of chitinase and extracellular protease were induced in the aphid after treatment with HK-1. A bioassay on A. craccivora showed that the median lethal concentration (LC50) of the fungus crude extract was 24.00 mg mL⁻¹ for 24 h of treatment. Additionally, the results showed that the crude extract disrupted the enzyme system of A. craccivora, inducing the inhibition of carboxylesterase (CarE) and the induction of glutathione S-transferase (GST) and acetylcholinesterase (AChE). Combining these results with those of a gas chromatography–mass spectrometry (GC-MS) analysis, it is suggested that p-cymene, hymecromone, 9,12-octadecadienoic acid (Z, Z) methyl ester, and 9,12-octadecadienoic acid (Z, Z) may be connected to the insecticidal effects we observed. This study provides a theoretical basis for the use of L. araneicola HK-1 as a potential biological control agent.
... Carbosulfan inhibits cholinesterase (ChE) and prevents the acetylcholine breakdown into acetic acid and choline, resulting in abnormal neurotransmission (Namba et al. 1971;Menozzi et al. 2004). Liposcelis bostrychophila (Psocop: Liposcelididae) is sensitive to carbosulfan (Cheng et al. 2004); Allium sativum, Azadirachta indica and C. deodara oils are toxic to this insect (Rao et al. 2003). Bio-insecticides affect glutamic pyruvic transaminase (GPT) and glutamic oxaloacetic transaminase (GOT), which are important biomarkers to detect cell rupture and cellular impairment (Mead-Hala 2000;Malarvizhi et al. 2012). ...
Specific compounds extracted from plants can control insect pests. The objective of this study was to evaluate the toxicity of deodar oil (phytopesticide) to adult mealworms Tenebrio molitor (Coleoptera: Tenebrionidae) compared with carbosulfan (synthetic insecticide), which exibits cholinesterase (ChE), glutamic pyruvic transaminase (GPT), and glutamic oxaloacetic transaminase (GOT) activities. The insecticides were applied through feeding, and the LC 50 (lethal concentration) was calculated using the Finney method. The LC 50 of deodar oil was higher than that of carbosulfan. The doses of both deodar oil and carbosulfan inhibited the ChE activity (p > 0.05) and enhanced the GPT and GOT activities (p < 0.05) in mealworm adults. Alterations in the activity of these biomarkers indicated that deodar oil could effectively control adult mealworms, being an environmentally low-impact method that can replace the use of chemical products.
... Carbosulfan inhibits cholinesterase (ChE) and prevents the acetylcholine breakdown into acetic acid and choline, resulting in abnormal neurotransmission (Namba et al. 1971;Menozzi et al. 2004). Liposcelis bostrychophila (Psocop: Liposcelididae) is sensitive to carbosulfan (Cheng et al. 2004); Allium sativum, Azadirachta indica and C. deodara oils are toxic to this insect (Rao et al. 2003). Bio-insecticides affect glutamic pyruvic transaminase (GPT) and glutamic oxaloacetic transaminase (GOT), which are important biomarkers to detect cell rupture and cellular impairment (Mead-Hala 2000;Malarvizhi et al. 2012). ...
Specific compounds extracted from plants can control insect pests. The objective of this study was to evaluate the toxicityof deodar oil (phytopesticide) to adult mealwormsTenebrio molitor (Coleoptera: Tenebrionidae) compared with carbosulfan (syntheticinsecticide), which exibits cholinesterase (ChE), glutamic pyruvic transaminase (GPT), and glutamic oxaloacetic transaminase (GOT)activities. The insecticides were applied through feeding, and the LC50 (lethal concentration) was calculated using the Finney method.The LC50 of deodar oil was higher than that of carbosulfan. The doses of both deodar oil and carbosulfan inhibited the ChE activity(p > 0.05) and enhanced the GPT and GOT activities (p < 0.05) in mealworm adults. Alterations in the activity of these biomarkersindicated that deodar oil could effectively control adult mealworms, being an environmentally low-impact method that can replacethe use of chemical products.
... [40] The mechanism of action of insecticide against L. bostrychophila was confirmed to be the inhibition of CarE and AChE. [41,42] The EOs from M. pteleifolia were proven to have insecticidal effects on storage pests. In this study, the plant samples were inexpensive (1.68 USD per kilogram), and the price is appropriate after the products have been developed into commodities. ...
Melicope pteleifolia is a traditional Chinese medicinal plant that is thought to be developed as a biological insecticide. In this work, essential oils (EOs) of M. pteleifolia (collected in 2021 and 2022) over different extraction time (1 h, 2 h, 5 h) were investigated for their chemical composition and insecticidal activities. The EOs extracted from fresh leaves in 2021 were named A1h, A2h and A5h, and extracted from fresh leaves in 2022 were named B1h, B2h and B5h. The chemical components of EOs were analyzed by GC-MS. α-Thujene is the common primary compound of the EOs A1h, A2h and A5h, which contained 83.27%, 63.86% and 25.17%, respectively. Principal component analysis (PCA) revealed that sesquiterpenes were the primary representative components in EOs. The EOs that were extracted for 5 h were significantly different from other EOs. Moreover, the EOs and α-thujene showed obvious contact and repellent activities against Lasioderma serricorne and Liposcelis bostrychophila. The gray correlation degree explained that EOs A2h and A5h had the strongest repellency against L. serricorne, while EOs A1h and B1h had the most significant repellent effect on L. bostrychophila, with coefficients above 0.50. The comprehensive score of weighting coefficient revealed that the scores of EOs A1h and A5h were the highest and both reached 0.80. In general, M. pteleifolia have the potential to be exploited as biological insecticide.
... Insecticide bioassays were conducted following the methods in our previously published work (Cheng et al., 2004), with a slight modification. Three insecticides, deltamethrin (pyrethroid; Sigma Aldrich, St. Louis, MO), malathion (organophosphorus; ChemService, West Chester, PA), and propoxur (carbamate; Sigma Aldrich), were used in this study. ...
In this study, the cDNAs of five cytochromes P450 genes (named CYP345P1, CYP358B1, CYP4FD2, CYP4CD2, and CYP6JN1) contained open reading frames from 1,500 to 1,554 nucleotides that encoded 499 to 517 amino acids were cloned from the psocid Liposcelis entomophila. They are characterized by predicted molecular weights from 57.67 to 59.64 kDa and theoretical isoelectric points of 5.57-9.07. Quantitative real-time PCR analysis showed these five genes were expressed at all tested developmental stages and higher expressions were observed in adults. CYP358B1 was expressed at higher levels in egg and adult compared to the larval stages. mRNA abundances of five genes were detected in both sexes and were relatively more abundant in adult females than in adult males. Synergism bioassay showed that the synergic ratio was 2.20 and 2.45 when insects were treated with the mixture of deltamethrin or malathion with the synergist piperonyl butoxide (PBO). Because PBO induces cytochrome P450s in some insects, this suggested to us that cytochromes P450 might participate in detoxification of these insecticides. The transcripts of the five cytochromes P450 genes in adult psocids could be induced to the highest level at 12 h after the exposure to malathion. After exposure to deltamethrin, CYP358B1 reached maximum expression at 24 h. The maximum expression of the other four genes occurred at 36 h. Treatments with the carbamate propoxur did not influence transcription of the cytochromes P450 gene. The induction profiles suggested that these five cytochrome P450 genes may be associated with deltamethrin and malathion metabolism in psocids.
... Among the detoxification enzymes, AChE is a key enzyme that terminates nerve impulses by catalyzing the hydrolysis of the neurotransmitter acetylcholine in the nervous system (Cheng et al., 2004). So, treatment with rosaside showed the lowest activity in AchE which resulted in its high efficacy in controlling aphid. ...
The current work aims to study the insecticidal effect of some compounds prepared as essential oil
formulations (rosacide, sagix and cura) against phloem sucking aphid (Aphis craccivora Koch) treated
systematically (soil treatment) or by spray (contact treatment). Results revealed that spray treatment was
most effective than systemic one, since the cumulative percentage mortality after 7 days were 93.33, 63.33
and 49.98 at 1% concentration for rosacide, sagix and cura respectively. The descending order of toxicity was
Rosacide > Sagix > Cura. Mode of action of the three tested compounds were examined by determination the
detoxification enzymes in aphid Glutathione S-transferase (GST) and acetylcholinesterase (AchE). Significantly
lower activities of both enzymes than control was found. The side effect of the three tested compounds on
the natural enemies Rodolia cardinalis on the aphid was studied. Data indicated that rosacide have moderate
effect on R. cardinalis larvae followed by (Sagix) and can overcome this effect by choosing the suitable time of
application in field when natural enemies with low density. Concerning the side effect of the tested compounds
on faba bean plants, Antioxidant activities of total phenol contents and GST had been investigated. The results
indicated that the treatment induced promising effect on the antioxidant enzymes activity. Also, photosynthetic
pigments have been studied. Data showed that there were no significant differences between treatment and
control. Moreover, the side effect of the tested compounds on the germination and shoot length of treated
bean seeds revealed that, there were no significant differences between the percentage of germination and
shoot length when the seeds treated with the tested compounds and control.
... In the last two decades, a series of papers about psocids have been published about their bionomics (Dong et al., 2007;Jiang et al., 2008;, ecology , control measures (Wang et al., 2001), and molecular biology Dong et al., 2007;Jiang et al., 2008;Tang et al., 2009) by Key Laboratory of Entomology and Pest Control of Chongqing Municipality (Southwest University, China). The main differences between L. bostrychophila and Liposcelis entomophila (Enderlein) about biochemical and toxicological characteristics of detoxification, protection, target enzymes and energy sources were clarified Cheng et al., 2004;. The activity of carboxylesterase (CarE) extracted from L. entomophila was significantly higher than that from L. bostrychophila, while for acid phosphatase (ACP), alkaline phosphatase (ALP) and glutathione Stransferases (GSTs) (Cheng et al., 2007), L. bostrychophila had more activities. ...
During the past several decades, China has made great progress in grain storage research, which greatly improved and promoted grain storage techniques and facilities. Especially in the 1990s, modern grain storage depots were constructed, in which four new technologies of grain inspection automation, machinery aeration, grain cooling, and phosphine recirculating fumigation were popularized. The main technologies to phase out methyl bromide in national depots are under-film phosphine recirculation fumigation and mixing fumigation of phosphine and carbon dioxide. In grain storage scientific research, research programs from applied to molecular fields have been implemented in institutes and universities. The 4th national survey of stored grain insects showed 270 species including 44 natural enemies were recorded. Among these stored grain insect pests the psocid, Liposcelis bostrychophila is generally more prevalent in stored grain with high moisture content. Hence, bionomics, ecologies, control measures, and molecular biology have being conducted since 1990s, and many achievements have been obtained. Efficacy of new grain protectants such as diatomaceous earth and spinosad against the main stored grain insects were evaluated. Researches on effective concentration of phosphine, application technique, recirculation fumigation, combination phosphine with carbon dioxide, and phosphine resistance mechanism were executed. Other fumigants tested included ethyl formate, carbon disulfide, and sulfuryl fluoride against stored grain insects were evaluated, or reevaluated. The fumigation activities and mechanisms of ethyl formate to the main stored product insects were systemically studied and the results showed ethyl formate controlled Sitophilus oryzae, Tribolium castaneum, Rhyzopertha dominica and Liposcelis bostrychophila effectively in a very short time, and the fumigation efficacy at relatively low temperature was better than that at relatively higher temperature. With the rapid progress of technology and scientific research, the objectives of high quality, high nutrition, high benefit, low waste, low pollution, and low cost in grain storage will be achieved, even during prolonged storage.
... For psocids, a series of studies have been reported in terms of biochemical characteristics of detoxifying enzymes and target enzymes from homogenate aspects [18][19][20][21]30]. Our previous study on homogenate GSTs has revealed that in L. bostrychophila there might be some correlation between the development of resistance and the quantity of GSTs [18]. ...
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.
... A bioassay using the pesticide, deltamethrin (analytical grade, East Sichuan Pesticide Factory, China), was conducted following previously described methods (Cheng et al. 2004) with slight modification. In brief, deltamethrin created a series of dilutions in acetone. ...
A β-actin gene, Libβ-actinl, from the psocid, Liposcelis bostrychophila, was isolated, sequenced, and expressed in Escherichia coli. The cDNA sequence was 1 281 bp in length and contained an open reading frame of 1 131 bp encoding 376 amino acids with a predicted molecular weight of 41.82 kDa. According to a BlastN search, the coding region shared the highest identity (97%) with Pediculus humanus actin 5C, while the deduced amino acid sequence was completely identical to a mutant of Drosophila melanogaster actin 5C. Comparison of the nucleotide and deduced amino acid sequences confirmed the high similarity between Libβ-actinl and homologs in other insect species. The 3′ untranslated region (3′ UTR) of the Libβ-actinl mRNA had a high A+U content (approximately 75%) and contained three repeats of the AUUUUUA and AUUUA motifs, which may play a role in regulating mRNA decay. The expression of Libβ-actinl was further analyzed in insecticide induced and control psocids. The results indicated that there was no significant difference in expression of Libβ-actinl between the induced and control groups, suggesting that Libβ-actinl may be an appropriate internal control for the gene expression profiling in this insect. Furthermore, Libβ-actinl was also heterologously expressed in Escherichia coli, which provided a basis to investigate the physiological functions of actin genes in the psocid.
... Elevated detoxification enzyme activities in insect midgut and fat body tissues are often associated with enhanced detoxification of allelochemicals (Brattsten and Wilkinson 1997) and/or insecticides (Valles et al. 1999). Among the detoxification enzymes, AChE is a key enzyme that terminates nerve impulses by catalyzing the hydrolysis of the neurotransmitter acetylcholine in the nervous system (Cheng et al. 2004). CarE catalyzes the hydrolysis of a wide variety of ester and amide containing endogenous and xenobiotic compounds. ...
To reveal the multi-generation effects of transgenic Bt cotton on the metabolism of secondary target herbivores, the activities
of digestive and detoxification enzymes in beet armyworm, Spodoptera exigua (Hübner), fed with transgenic Bt (cv. GK-12) vs. non-Bt cotton (cv. Simian-3) for three successive generations were examined.
Significantly lower activities of lipase, carboxylesterase (CarE) and acetylcholinesterase (AChE), and higher activities of
trypsin and total superoxide dismutase (T-SOD) were observed in Bt-fed S. exigua than in non-Bt fed individuals during the three generations. But the activity of amylase did not differ between the Bt-fed
and non-Bt fed S. exigua. Among the three generations of Bt-fed S. exigua, the activities of lipase and trypsin decreased significantly in the second or third generation than in the first generation.
But those of CarE and AChE were increased significantly. Among the three generations of non-Bt fed S. exigua, the activity of lipase was significantly higher in the second and third generations than in the first generation. The increasing
adaptation and fitness of S. exigua after continuous exposure to Bt cotton was supposed to be associated with the enhanced activities of detoxification enzymes.
Measuring multi-generational enzymes activities of S. exigua in response to Bt cotton can provide proofs of its adaptation development, which is suggested to be a mandatory part of risk
assessment of transgenic Bt plants on secondary target herbivores.
Keywords
Spodoptera exigua
-Transgenic Bt cotton-Ecological risk assessment-Multi-generation-Detoxification enzyme-Digestive enzyme
... To date, the research on the psocids continues to increase but the knowledge is still quite limited compared to other stored-product insect pests. Our previous study has provided some important information about crude preparations of detoxifying enzymes (GSTs included) and target enzymes in the psocids (Wang et al. 2004; Cheng et al. 2004 Cheng et al. , 2007 Dou et al. 2006; Chai et al. 2007; Ren et al. 2008). Recently, the GSTs from L. bostrychophila and L. paeta was purified, respectively, and their partial characterizations were subsequently analyzed from the perspective of resistance of the different strains or field populations (Dou et al. 2009; Wu et al. 2009 ). ...
Glutathione S-transferases (GSTs) from Liposcelis bostrychophila Badonnel and L. entomophila (Enderlein) (Psocoptera: Liposcelididae) were purified by glutathione-agarose affinity chromatography, and characterized subsequently by their Michaelis-Menten kinetics toward the artificial substrates 1-chloro-2,4-dinitrobenzene (CDNB) and reduced glutathione (GSH), respectively. The specific activity of the purified GST toward CDNB was 2.3-fold higher in L. bostrychophila than in L. entomophila. Though the specific activities of purified enzymes varied between the two species, the purification yields were similar. SDS-PAGE revealed one band at 23 kDa for both the species. GSTs of L. entomophila exhibited higher Michaelis-Menten constants (Km) but lower maximal velocity (Vmax) values than those of L. bostrychophila. The optimum pH for CDNB conjugation of L. bostrychophila and L. entomophila GSTs was 7.0 and 7.5, and optimum temperature was 35 and 40°C, respectively. Inhibition kinetics showed that cibacron blue, curcumin, bromosulfalein, ethacrynic acid, and carbosulfan had excellent inhibitory effects on GSTs in both species, but the inhibitory effects of beta-cypermethrin, fenpropathrin, tetraethylthiuram disulfide, and diethyl maleate were not significant.
... Deltamethrin (analytical grade, East Sichuan Pesticide Factory, China) exposure was conducted following the previous published methods (Cheng et al., 2004) with slight modification. In brief, deltamethrin was diluted to 2.5 mg/L in acetone. ...
Two novel P450 genes, CYP6CE1 and CYP6CE2 (GenBank accession number: EF421245 and EF421246), were cloned and characterized from psocid, Liposcelis bostrychophila. CYP6CE1 and CYP6CE2 contain open reading frames of 1,581 and 1,563 nucleotides that encode 527 and 521 amino acid residues, respectively. The putative proteins of CYP6CE1 and CYP6CE2 show predicted molecular weights of 60.76 and 59.83 kDa with a theoretical pI of 8.58 and 8.78, respectively. CYP6CE1 and CYP6CE2 share 74% identity with each other, and the deduced proteins are typical microsomal P450s sharing signature sequences with other insect CYP6 P450s. Both CYP6CE1 and CYP6CE2 share the closest identities with Hodotermopsis sjoestedti CYP6AM1 at 48% among the published sequences. Phylogenetic analysis showed a closer relationship of CYP6CE1 and CYP6CE2 with CYP6 members of other insects than with those from other families. Quantitative real-time RT-PCR showed that both CYP6CE1 and CYP6CE2 are expressed at all developmental stages tested. Interestingly, CYP6CE2 transcripts decreased from the highest in 1st nymph to the lowest in adults, which seemed to suggest developmental regulation. However, neither CYP6CE1 nor CYP6CE2 were stage specific. The CYP6CE1 and CYP6CE2 transcripts in adults increased significantly after deltamethrin exposure. Recombinant protein expression studies are needed to determine the real functions of these proteins.
... A comparative analysis of substrate-and inhibitor-induced modulation of CbE activity with other related studies reveals that this esterase can play a determinant role in the intestinal pesticide uptake by earthworms. Gut CbE activity of L. terrestris exhibited a high affinity for the substrates used in this study (K m 's values ranging between 75 µM for α-NA and 204 µM for 4-NPA) compared, for instance, to CbE activity of the terrestrial snail Xeropicta derbentina (K m 's values ranging between 222 µM for α-NA and 980 µM for 4- NPA) (Laguerre et al., 2009), or those reported for the insect Liposcelis entomophila (K m = 280 µM for α-NA) and Liposcelis bostrychophila (K m = 660 µM for α-NA) (Cheng et al., 2004). Furthermore, the catalytic efficiency of CbE increased largely in the earthworm gut content compared to that exhibited by tissue CbE. ...
Carboxylesterases (CbEs) are key enzymes in pesticide detoxification. These esterases are involved in the biochemical mechanism for pesticide resistance in some pest species, and further they are considered an efficient protective mechanism against acute toxicity by organophosphate (OP) pesticides in mammals. To gain knowledge on the role of CbEs in pesticide toxicity and natural tolerance in earthworms, we performed an enzyme kinetic analysis to investigate whether these annelids are able to secrete them into their gut lumen. We determined levels of CbE activity and isozyme abundance in the gut wall and ingested soil collected from different portions of the gastrointestinal tract of Lumbricus terrestris. Moreover, modulation of enzyme activity by selected substrates (alpha-naphthyl acetate [alpha-NA], 4-nitrophenyl valerate [4-NPV] and 4-nitrophenyl acetate [4-NPA]) and OP pesticides was examined to compare the response between tissue and soil CbEs. We found a high CbE activity in the ingested soil extracts from the crop/gizzard (alpha-NA-CbE=8.43+/-2.76U mg(-1) protein and 4-NPA-CbE=5.98+/-2.11U mg(-1) protein) compared to the gut wall. Three lines of evidences suggest that the gut epithelium is the main source of this luminal CbE activity. First, the effect of substrate concentrations on CbE activity from both the ingested soil extracts and gut tissues resulted in similar apparent K(m) and V(max) values. Second, native PAGE gels revealed that some of the CbE isozymes in the gut tissue were also present in the soil extracts. Third, tissue and soil CbEs showed the same sensitivity to inhibition by OPs. The concentrations of insecticide causing 50% of esterase inhibition (IC(50)) was comparable between tissue (IC(50)s range=4.01-9.67nM dichlorvos and 8480-6880nM paraoxon) and soil (IC(50)s range=6.01-11.5nM dichlorvos and 8400-7260nM paraoxon). Our results suggest a set of (eco)toxicological implications and environmental applications derived from the ability of earthworms to secrete these pesticide-detoxifying enzymes.
Esterases (ESTs) play important roles in metabolizing various physiologically endogenous and exogenous compounds, and various environmental xenobiotics in insects. The psocid, Liposcelis bostrychophila is a major pest of stored products worldwide and rapidly develops resistance to commonly insecticides. However, the involvement of ESTs in insecticide metabolization and the application of RNAi approach in psocids have not been well elucidated. In this study, we characterized four LbEST genes and investigated the transcriptional levels of these genes at different developmental stages and under different insecticides exposures to assess their potential roles in response to insecticides. The four LbESTs contain a catalytic triad (Ser-His-Glu) linked to an oxyanion hole and acyl pocket involved in substrate stabilization during its hydrolysis. Synergism observed with the esterase-inhibitor DEF suggests the involvement of esterases in malathion detoxification. LbESTs were expressed during the whole of developmental stages, but predominant abundance in the first nymphal instar and adult stage. The mRNA level of three LbEST genes (except for LbEST4) was induced (1.29- to 5.60 fold) in response to malathion or deltamethrin exposures, indicating that these esterases are involved in the detoxification process. Silencing of LbEST1, LbEST2 or LbEST3 through dsRNA feeding led to a higher mortality of psocids upon the malathion treatment compared to controls (1.83 to 2.69-fold), demonstrating that these esterase genes play roles in malathion detoxification in L. bostrychophila. Our study provides new evidence for understanding of the function and regulation mechanism of esterases in L. bostrychophila in insecticide detoxification. The current study also suggests that the present RNAi method could be applied for gene functional studies in psocids.
We investigated the effects often constant temperatures (20.0, 22.5, 25.0, 27.5, 30.0, 32.5, 35.0, 37.5, 39.0, and 41.0 degrees C) on the development, survival, and reproduction of the psocid Liposcelis yunnaniensis Li & Li (Psocoptera: Liposcelididae). At 39.0 and 41.0 degrees C, none of individuals could develop successfully or reproduce. From 20 to 37.5 degrees C, the development period from egg to adult ranged from 64.3 d at 20 degrees C to 16.1 d at 35 degrees C. The lower developmental threshold for egg, nymph, and combined immature stages were estimated at 15.08, 15.13, and 14.77 degrees C, respectively. After emergence the females went through a preoviposition period that ranged from 18.5 d at 22.5 degrees C to 3.11 d at 35 degrees C, whereas it was 16.3 d at 20 degrees C. Liposcelis yunnaniensis produced most eggs at 35 degrees C and the fewest at 22.5 degrees C. The population reared at 35 degrees C had the highest intrinsic rate of increase, shorter mean generation time, and shortest population doubling time compared with other temperatures. According to Weibull frequency distribution, L. yunnaniensis reared at all the temperatures had type III survivorship curves (c < 1.0). Based on life-table parameter estimations, we suggest that optimum range of temperatures for this species is from 25 to 37.5 degrees C. These data give us useful information on population biology of L. yunnaniensis and can be used to better manage this species.
The development, survival, and reproduction of Liposcelis decolor (Pearman) (Psocoptera: Liposcelididae), an important insect pest of infested stored products, were evaluated at eight constant temperatures (20-37.5 degrees C). Female L. decolor had four nymphal stadia, whereas males only had three nymphal stadia. Between 20 and 37.5 degrees C, the female developmental period from egg to adult varied from 46.2 d at 20 degrees C to 16.1 d at 35 degrees C, and the male developmental period from egg to adult varied from 41.8 d at 20 degrees C to 13.6 d at 35 degrees C. Based on a nonlinear model, the lower temperature developmental thresholds of female from egg, first through fourth stadia, and combined immature stages were estimated at 12.22, 16.08, 10.93, 8.88, 12.64, and 13.02 degrees C, the upper temperature thresholds were 42.11, 39.44, 41.27 40.92, 39.20, and 40.52 degrees C, respectively, whereas the lower temperature thresholds of males ranged from 11.68 to 15.68 degrees C, and the upper temperature thresholds ranged from 40.19 to 42.04 degrees C. The survival rate from egg to adult was 57.3% at 32.5 degrees C, 38.4 at 20 degrees C, and 19% at 37.5 degrees C, respectively. After emergence, the adult had a preoviposition period that ranged from 6.8 d at 20 degrees C to 2.3 d at 35 degrees C. L. decolor produced the most eggs (130.4) at 32.5 degrees C and the fewest (24.7) at 37.5 degrees C. The population reared at 32.5 degrees C had the highest intrinsic rate of increase (0.0609) compared with the populations reared at seven other constant temperatures. The populations reared at 35 and 37.5 degrees C had type III survivorship pattern, whereas populations reared at other temperatures had type I survivorship curve as determined by a Weibull frequency distribution. The optimal range of temperature for L. decolor population growth was 27.5-35 degrees C.
Liposcelis tricolor Badonnel (Psocoptera: Liposcelididae) is worldwide and commonly found in various processed and unprocessed dry foods in households, granaries, and warehouses. The development, survival, and reproduction of L. tricolor were evaluated at eight constant temperatures (20, 22.5, 25, 27.5, 30, 32.5, 35, and 37.5°C). The population reared at 37.5°C failed to develop or reproduce. Between 20 and 35°C, developmental period from egg to adult varied from 56.37 d at 20°C to 30.74 d at 32.5°C. Based on a nonlinear model, the lower temperature developmental thresholds from egg, first through fourth stadia, and combined immature stages were estimated at 9.57, 9.02, 10.04, 13.69, 14.38, and 11.30°C; the upper temperature thresholds were 39.28, 37.55, 37.85, 38.26, 37.37, and 38.98°C, respectively. The survival rate from egg to adult was 67.26% at 27.5°C, 43.62 and 24.22% at 20 and 35°C, respectively. After emergence, the adult had a preoviposition period that ranged from 65.74 d at 20°C to 3.52 d at 30°C. L. tricolor produced the most eggs (53.91) at 27.5°C and the fewest (17.58) at 35°C. The population reared at 30°C had the highest intrinsic rate of increase (0.0367), net reproductive rate (13.55), the shortest populations doubling time (18.89 d), and shorter mean generation time (71.05 d) compared with the populations reared at six other constant temperatures. The populations reared at 30°C had type I survivorship pattern, whereas populations reared at other temperatures had type III survivorship curve as determined by a Weibull frequency distribution. The optimal range of temperature for L. tricolor population growth was 25–30°C.
The cDNAs of three novel P450 genes, CYP4CB1, CYP4CC1, and CYP4CD1 (GenBank accessions EU979550, EU979549, and EU979551, respectively), were sequenced and characterized from the psocid Liposcelis bostrychophila Badonnel (Psocoptera: Liposcelididae). CYP4CB1, CYP4CC1, and CYP4CD1 contain open reading frames of 1533, 1512, and 1536 nucleotides that encode 511,504, and 512 amino acid residues, respectively. The putative proteins of CYP4CB1, CYP4CC1, and CYP4CD1 show predicted molecular mass of 59.65, 58.87, and 59.71 kDa, with a theoretical isoelectric point of 6.59, 8.80, and 8.84, respectively. The N-terminal transmembrane domain was only found in CYP4CB1 suggested it is a typical microsomal P450. Phylogenetic analysis showed a close relationship of CYP4CB1, CYP4CC1, and CYP4CD1 with CYP4AW1, CYP4L4, and CYP4E2. Quantitative real-time reverse transcriptase-polymerase chain reaction indicated that these three genes were expressed at all tested developmental stages. In addition, the highest expression occurred in the adult stage, which suggested that these three P450 genes may play important roles in adulthood. The transcripts of CYP4CB1 and CYP4CC1 in adult psocids could be induced to the highest level at 36 and 24 h after the exposure to deltamethrin and paraoxon-methyl (50% lethal concentration [LC50]), respectively, whereas CYP4CD1 remained unchanged. CYP4CD1 transcripts, however, increased rapidly at 8 h after aldicarb (LC50) induction and reached the peak at 36 h. The induction profiles of the three P450 genes suggested that CYP4CB1 and CYP4CC1 are possibly associated with deltamethrin and paraoxonmethyl metabolism in psocids, whereas CYP4CD1 is probably involved in aldicarb metabolism. However, our assumption needs to be further verified by recombinant protein expression of these proteins as well as RNA interference of these genes.
The toxicological and biochemical characteristics of acetylcholinesterases (AChE) in Liposcelis paeta Pearman were investigated in three field populations collected from Nanyang city of Henan Province (NY), Wuzhou (WZ) and Hezhou (HZ) Cities of Guangxi Province, China. The result of bioassay showed that the LC50s of the NY (281.4802 mg/m2) and the WZ (285.0655 mg/m2) to dichlorvos were 1.156-fold and 1.171-fold higher than that of the HZ (243.5197 mg/m2), respectively. Compared to NY population, the activity per insect and the specific activity of AChE in WZ and HZ populations were significantly higher, and significant kinetic differences among the three populations were also observed. The apparent Michaelis–Menten constant (Km) for acetylthiocholine iodide (ATChI) was obviously lower in NY than that in WZ and HZ populations, indicating a higher affinity to the substrate ATChI in the NY population. The affinity to the substrate ATChI between WZ and HZ population was also significantly different. As for Vmax, the values of WZ and HZ populations were significantly greater when compared to that for NY population, suggesting a possible over expression of AChE in the former two populations. The inhibition studies of AChE indicated that paraoxon-ethyl, demeton-S-methyl, carbaryl, and eserine all possessed some inhibitory effects on AChE in L. paeta. The results of I50S suggested that when compared to the other two populations, while AChE from HZ population was less sensitive to paraoxon-ethyl and demeton-S-methyl. The contradiction with the result of the bioassay might be due to the different insecticides used in the bioassay. Although both carbaryl and eserine had excellent inhibitory effects, there was no significant difference among the three populations. The statistical analysis of the bimolecular rate constants (ki) was consistent with the above situation that carbamates expressed remarkable inhibitory effects. It was noticeable that NY population was most sensitive to carbaryl while least to eserine. The differences in AChE among three populations may attribute to the difference in control practices for psocids between Henan and Guangxi Provinces.
Acetylcholinesterace (AChE) is known to be the major target for organophophate and carbamate insecticides and biomolecular changes to AChE have been demonstrated to be an important mechanism for insecticide resistance in many insect species. In this study, AChE from three field populations of Liposcelis entomophila (Enderlein) (Psocoptera: Liposcelididae) was purified by affinity chromatography and subsequently characterized by its Michaelis-Menten kinetics to determine if detectable changes to AChE have occurred. Bioassays revealed that the potential resistance threat of psocids in Sichuan Province (GH) was greater than either Hubei Province (WH) or Chongqing Municipality (BB). Compared to the other two populations, the WH population possessed the highest specific activity of purified AChE. Kinetic analyses indicated that the purified AChE from GH population expressed a significantly lower affinity to the substrate and a higher catalytic activity toward acetylthiocholine iodide (ATChI) (i.e., higher K(m) and V(max) values) than BB and WH populations. In vitro studies of AChE suggest that five inhibitors (aldicarb, eserine, BW284C51, omethoate, and propoxur) all possess strong inhibitory effects with eserine having the strongest inhibitory effect against purified AChE. According to bimolecular rate constants (k(i)), the purified AChE from GH population was least sensitive to all inhibitors except for omethoate. The differences in AChE among the three populations may be partially attributed to the differences in pesticide application and control practices for psocids among the three locations.
The nutrient composition and enzyme activities in larvae of the beet armyworm, Spodoptera exigua (Hübner), fed on high, medium or low gossypol cotton cultivars were examined at different time intervals. Significantly lower free fatty acid was observed in larvae fed for 6 h on high gossypol 'M9101' compared to larvae fed on the low (ZMS13) and intermediate (HZ401) gossypol cultivars. Significantly higher trypsin activity was observed in larvae fed on high gossypol 'M9101' for 24 h compared to those fed for 1, 4 and 6 h. Significantly higher catalase and total superoxide dismutase enzyme activities were observed in larvae of S. exigua fed on high gossypol 'M9101' compared with low gossypol cultivars 'ZMS13' and 'HZ401' for 1, 4, 6 and 24 h. However, significantly lower carboxylesterase and acetylcholinesterase enzyme activities were found in larvae fed on high gossypol 'M9101' compared with the other cultivars for 1, 4, 6 and 24 h. The interaction between cotton variety and beet armyworm infestation time significantly affected the carboxylesterase enzyme activity in S. exigua. The characterization of the effects of plant allelochemicals on herbivorous larvae is important for aiding understanding of plant-insect interaction as well as in devising solutions to pest problems by breeding plant resistance, identifying metabolic targets for insecticide development, etc.
Psocids (Liposcelis spp.) are often abundant in tropical storage systems where very large populations can develop relatively soon after fumigation treatments. In order to improve pest management strategies against these pest some factors governing the potential of psocid populations to increase under tropical conditions were investigated in a large‐scale milled rice store and in the laboratory. The factors studied included the success of pest control, rice milling degree, fungal growth and variations in ambient temperature and humidity. Fumigation failure and low milling degree of the rice stock were confirmed as important factors encouraging rapid and substantial psocid population growth while fungal infection rates, routine spraying of store and bag stack and variations in ambient climatic conditions made no obvious contribution to observed fluctuations in psocid numbers. Suggestions are made for improved pest management strategies that need to be tested under operational conditions.
Esterase activities associated with organophosphate insecticide resistance in the Australian sheep blowfly, Lucilia cuprina, are compared with similar activities in other Diptera. The enzymes making the major contribution to methyl butyrate hydrolysis (ali-esterase) in L. cuprina, M. domestica, and D. melanogaster comigrate during electrophoresis. The enzymes in L. cuprina and D. melanogaster correspond to the naphthyl acetate hydrolyzing E3 and EST23 isozymes of those species. These and previously published data suggest that the ali-esterases of all three species are orthologous. Strains of L. cuprina fall into four groups on the basis of quantitative determinations of their ali-estesterase, OP hydrolase, and malathion carboxylesterase activities and these groups correspond to their status with respect to two types of OP resistance. Strains susceptible to OPs have high ali-esterase, low OP hydrolase, and intermediate MCE activities; those resistant to malathion but not diazinon have low ali-esterase, intermediate OP hydrolase, and high MCE activities; those resistant to diazinon but not malathion have low ali-esterase, high OP hydrolase, and low MCE activities; those resistant to both OPs have low ali-esterase, high OP hydrolase, and high MCE activities. The correlated changes among the three biochemical and two resistance phenotypes suggest that they are all properties of one gene/enzyme system; three major allelic variants of that system explain OP susceptibility and the two types of OP resistance. Models are proposed to explain the joint contribution of OP hydrolase and MCE activities to malathion resistance and the invariant association of low ali-esterase and elevated OP hydrolase activities in either type of resistance.
The enzyme, esterase B2, involved in insecticide resistance has been purified and characterized from the mosquito Culex quinquefasciatus. The monomeric enzyme has an M(r) of 62000 and a pI of 5.0. This enzyme is compared with the esterase A2 previously characterized [Ketterman, Jayawardena and Hemingway (1992) Biochem. J. 287, 355-360]. The kinetic constants for interaction with several insecticides indicate, as for the esterase A2, that the B2 enzyme has a role in resistance. The rates and affinities of binding observed support the hypothesis that the role mainly is sequestration followed by the slow turnover of the insecticide. Although the B2 esterase appears to have a slightly higher rate of interaction with insecticides, the A2 has a much greater Vmax. with the xenobiotic substrates studied. The B2 esterase also appears to be present in the larvae to a lesser extent than the esterase A2.
Resistance to organophosphorus (OP) insecticides is associated with decreased carboxylesterase activity in several insect species. It has been proposed that the resistance may be the result of a mutation in a carboxylesterase that simultaneously reduces its carboxylesterase activity and confers an OP hydrolase activity (the "mutant ali-esterase hypothesis"). In the sheep blowfly, Lucilia cuprina, the association is due to a change in a specific esterase isozyme, E3, which, in resistant flies, has a null phenotype on gels stained using standard carboxylesterase substrates. Here we show that an OP-resistant allele of the gene that encodes E3 differs at five amino acid replacement sites from a previously described OP-susceptible allele. Knowledge of the structure of a related enzyme (acetylcholinesterase) suggests that one of these substitutions (Gly137 --> Asp) lies within the active site of the enzyme. The occurrence of this substitution is completely correlated with resistance across 15 isogenic strains. In vitro expression of two natural and two synthetic chimeric alleles shows that the Asp137 substitution alone is responsible for both the loss of E3's carboxylesterase activity and the acquisition of a novel OP hydrolase activity. Modeling of Asp137 in the homologous position in acetylcholinesterase suggests that Asp137 may act as a base to orientate a water molecule in the appropriate position for hydrolysis of the phosphorylated enzyme intermediate.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
Adult populations of the psocid, Liposcelis bostrychophila , were exposed for 30 generations to 35% CO2, 1% O2, and 64% N2 at 28oC, 80% RH, in order to select a strain resistant to controlled atmosphere (CA). Selection pressure was maintained at around 70% mortality. At the 30th generation, comparison of sensitivity between the selected strain and the original susceptible strain (CA-S) indicated a tolerance factor (TF) at the 50% mortality level (LT50) of 5.6-fold. Throughout the selection process, log-time against probit-mortality lines remained roughly parallel and the slopes remained lower than that of CA-S until the last generation. Implications are that at high levels of selection, multiple genetic factors continued to act together even at the 30th generation to select for adaptation to survival in a CA environment. Removal of selection pressure for five generations from one subpopulation of the selected strains from the 25th generation caused significant reduction in resistance. In the absence of CA exposure, the selected strain possessed the fitness defect. The CA-selected strain was calculated by R0 to have a fitness value of 0.39 relative to CA-S. In addition, the potential effective measures in the slowdown of resistance to CA of stored-product insects are also discussed.
Over the last 30 years the booklouse, Liposcelis bostrychophila has become an increasingly common domestic pest of stored food products. This review draws together published and unpublished information on the biology, population dynamics, and physiology of this species. The current pest status of L. bostrychophila in the UK and elsewhere is described. Emphasis is given to the parthenogenetic nature of reproduction and the surprising degree of variability seen in populations from different locations in the UK. A variety of approaches to the management of booklice as pests are explored, both in the industrial and domestic setting. Tolerance to pyrethroid insecticides has been found in some populations and is identified as a potential problem in current control practices.
Carboxylesterase E4, the enzyme previously shown to cause resistance to organophosphorus insecticides in peach-potato aphids, Myzus persicae, was purified and characterized by electrophoretic and enzyme kinetic techniques. Its insecticidal substrate specificity, determined by following the rate of recovery of esterase activity after inhibition by a range of acylating inhibitors, included a variety of carbamates and organophosphates, although the catalytic center activity for these substrates was low. Radiometric measurement of hydrolysis of the pyrethroid, permethrin, showed that E4, whether purified or in crude aphid homogenates, hydrolyzed the (1S)trans enantiomer rapidly but hydrolysis of the other three isomers could not be detected. Such absolute specificity for one enantiomer of a pyrethroid is rare. The rates of hydrolysis of the various insecticidal classes correlated well with the relative degrees of resistance to them, and no other resistance mechanisms have been detected. Although the enzyme is relatively inefficient in degrading insecticidal esters, it is produced in very large quantity, accounting for approximately 3% of the total protein in very resistant aphids. Its effect is thus mediated not only by hydrolysis but also by sequestering a substantial proportion of a toxic dose of insecticide. It is effective in this respect because the molar amount present is similar to that of a lethal dose of insecticide. These results support earlier indirect evidence for “overproduction” of E4, probably because of structural gene duplication or amplification and have direct implications for strategies to delay the buildup of resistance or for developing synergists to overcome resistance.
Eggs and adults of Liposcelis bostrychophila were exposed to fixed phosphine concentrations for varying exposure periods. The eggs were more tolerant of phosphine than the adults, but the former showed no change in tolerance with age. Phosphine may have arrested development of the eggs during exposure, as evidenced by delayed hatching of phosphine treated eggs. The response of L. bostrychophila eggs did not differ greatly from the simple relationship, Ct = k. When mixed-age cultures of L. entomophila were exposed to 0.1 mg I− 1 phosphine with varying exposure periods, the time to population extinction occurred at 96 h exposure. This dosage is similar to the LD99 for L. bostrychophila eggs at this concentration. The current recommendations for the use of phosphine are adequate for controlling the two species studied.
Methods for assaying AChE and non-specific esterase activity from Liposcelis spp. were outlined. Liposcelis bostrychophila AChE was less sensitive to inhibition than L. entomophila to malaoxon and DDVP. Non-specific esterases of L. bostrychophila were more susceptible to inhibition than those of L. entomophila. Totals of 10 and 14 esterase bands were detected on PAGE for L. bostrychophila and L. entomophila, respectively. The link between esterase sensitivity to inhibition and the differential insecticide tolerance between the two Liposcelis spp. is examined.
Organophosphate resistance in Culex mosquitos is commonly due to elevation of esterase activity. The usual form of elevation involves esterases A2 and B2 which are always together in the same individual insect. Using previously established purification methods, the purified esterases A2 and B2 from three resistant strains of Culex quinquefasciatus have been isolated and characterized. Substrate kinetic studies as well as the kinetics of insecticide interaction demonstrate that the esterases A2 and B2 differ both from each other and in the forms found in the three strains. That variations exist in the esterases in several resistant strains indicates polymorphism in the mosquito enzymes. This finding supports the hypothesis of insecticide resistance occurring multiple times and selecting different resistance-conferring esterases.
Strains of Lucilia cuprina (Wiedemann) have been characterized as having low, internlcdiate, or high levels of esterase-mediated hydrolysis of the organophosphorus insecticide, chlorfenvinphos. These levels correlate respectively with susceptibility to organophosphorus insecticides, malathion resistance, or diazinon resistance. Diazinon and chlorfenvinphos are diethyl organophosphorus insecticides having 2 methoxy groups attached to their central phosphorus atom, whereas malathion is a dimethyl organophosphorus insecticide having 2 methoxy groups attached to its phosphorus atom, and, unusually, malathion also has 2 carboxylester bonds in addition to the phosphoester bonds that define organophosphorus compounds. We tested larvae for resistance to diazinon and also assessed representative malathion-resistant and diazinon-resistant L. cuprina strains at the adult stage for resistance to 12 organophosphorus insecticides, including analog pairs differing only in respect to their dimethyl- diethyl status. Two malathion-resistant strains have low-level cross-resistance to diazinon (3 to 4-fold), 4 diazinon resistant strains have high-level diazinon resistance (11 to 16-fold), and 2 strains with a combined (malathion plus diazinon) resistance type also have high-level diazinon resistance (17 to 18-fold) relative to 3 organophosphorus insecticide-susceptible strains. One of the diazinon-resistant strains showed ≍ 2 times greater resistance factors toward diethyl organophosphorus insecticides than their dimethyl analogs while (leaving aside malathion to consider only the majority which have no carboxylester groups) a malathion-resistant strain showed 2-5 times greater resistance factors toward the dimethyl organophosphorus insectides than their diethyl analogs. The diazinon-resistant strain showed no resistance to 2 di-isopropyl organophosphorus compounds or to 2 organophosphorus insecticides which are asymmetric about the phosphorus atom (optically active). The malathion-resistant strain showed only slight resistance <3-fold) to either the di-isopropyl or optically active organophosphorus insecticides, including the di-isobropyl analog of malathion. These cross-resistance patterns parallel those of certain organophosphorus insecticide-resistant strains of Musca domestica L., in which diazinon and malathion resistances also are proposed to be esterase mediated, reinforcing other biochemical data suggesting a general mechanism among the higher Diptera
Laboratory assays were done to quantify proportions of phenotypes in laboratory colonies resistant to several classes of insecticides in the cotton aphid, Aphis gossypii Glover. Resistance to a carbamate (aldicarb), an organochlorine (endosulfan), an organophosphate (chlorpyrifos), and a pyrethroid (bifenthrin) was documented. Significant declines in numbers of individuals resistant to bifenthrin and endosulfan but not for chlorpyrifos occurred during a 5-mo interval after the first bioassays. Resistance ratios between clonal susceptible and resistant cotton aphid colonies for chlorpyrifos indicated a significant 4-fold difference in LCso estimates for the two colonies. Spectrophotometric analyses indicated Significantly higher carboxylesterase activity in resistant aphids compared with susceptible aphids. Results from isoelectric polyacrylamide focusing gel techniques also showed differences in carboxylesterases, both in differences in banding patterns and in expression of comigrating bands.
The role of esterases in dichlorvos resistance of the cotton aphid, Aphis gossypii Glover was studied by vertical slab polyacrylamide gel electrophoresis by using a susceptible (E-S) and dichlorvos-selected resistant (E-D-R) strain. Results suggested that both resistant and susceptible strains possessed bands E-2, E-11, and E-12, with the latter 2 bands of the resistant strain comigrating slightly faster compared with the susceptible strain. On addition, the resistant strain had additional bands (i.e., E-1, and E-3 to E-10), which probably reflected from changes in conformation. Although bands of both strains exhibited equal affinity (with the shorter naphthyl acetates and indoxyl acetate), none had affinity with naphthyl laurate and myristate. The most elevated in the resistant strain were E-7, E-9, and E-10, which were classified as carboxylesterases. Bands E-11 and E-12 in both strains were classified as eserine sulfate sensitive carboxylesterases. Excessive production of comigrating bands of carboxylesterases in both quantitative and qualitative forms, coupled with modification of these enzymes, may account for dichlorvos resistance in this aphid.
A method for the purification of elevated esterase B2 from the Sri Lankan Pel RR strain of Culex quinquefasciatus (Say) has been developed, using sequential column chromatography. The pure enzyme is stable, with no decrease in specific activity after several months, if stored in the presence of 25 mM dithiothreitol and 50% glycerol at –20°C. The enzyme has a pl of 5 and an estimated molecular weight for the monomeric enzyme of 63 kD from SDS-PAGE. In all Culex strains with elevated B2, another esterase (A2) is always co-elevated. The strains with these esterases are resistant to a broad range of organophosphorus insecticides. The previous purification and characterization of A2 from Pel RR indicated that the role of this enzyme was primarily to sequester rather than metabolize organophosphates. Purified B2, rapidly binds malaoxon, the activated form of malathion, but not malathion itself. The turnover rate of bound malaoxon is slow, suggesting that the role of this esterase in malathion resistance is also sequestration. The similar bimolecular rate constants for both A2 and B2 with malaoxon suggest both esterases are important in resistance, which may explain the almost complete linkage disequilibrium that the elevated forms of these two esterases are maintained in when under pesticide selection pressure.
Two populations of the psocid, Liposcelis bostrychophila Badonnel, were exposed to two CO2-enriched atmospheres (35% CO2 + 21% O2, and 55% CO2 + 21% O2, balance N2) for 30 generations. Controls were reared in normal atmospheres. The reserves of triacylglycerol and polysaccharides were evaluated in adults of the two experimental and the control populations in generations F15 and F30. The utilization rate of triacylglycerol and polysaccharides in the CO2-enriched atmospheres were also determined in generation F30. The results indicated that the reserves of triacylglycerol and polysaccharides increased significantly during selection for CO2 resistance; the higher the resistance level, the greater the reserves. Exposure of these populations to controlled atmosphere was associated with a steady utilization of the reserves. By contrast, the unselected population responded to controlled atmospheres by accelerated utilization of triacylglycerol and polysaccharides. Comparison of the utilization rates during CO2 exposure showed that triacylglycerol is the main energy source, and polysaccharides contribute to metabolic energy supply only to a small extent.
Two types of organophosphorus (OP) insecticide resistance are associated with reduced `ali-esterase' (E3 isozyme) activity in Lucilia cuprina. The `diazinon' resistance type shows generally greater resistance for diethyl than dimethyl OPs but no resistance to malathion. The `malathion' resistance type shows generally greater resistance for dimethyl than diethyl OPs, low level diazinon resistance, but exceptionally high malathion resistance (600 × susceptible), the last being attributed to hydrolysis of the carboxylester groups which are peculiar to malathion (malathion carboxylesterase, MCE). E3 variants from diazinon resistant strains have previously been shown to have a Gly137 → Asp substitution that structural modelling predicts is only about 4.6 Å from the γ oxygen of the catalytic serine residue. Here we show that E3 variants from malathion resistant strains have a Trp251 → Leu substitution predicted to be about 4.3 Å from that serine. We have expressed alleles of the gene encoding both resistance variants of E3 and an OP susceptible variant in a baculovirus system and compared the kinetics of their products. We find that both resistance substitutions reduce ali-esterase activity and enhance OP hydrolase activity. Furthermore the Gly137 → Asp substitution enhances OP hydrolase activity for a diethyl OP substrate (chlorfenvinphos) more than does the Trp251 → Leu substitution, which is consistent with the OP cross-resistance patterns. Trp251 → Leu also reduces the Km for carboxylester hydrolysis of malathion about 10-fold to 21 μM, which is consistent with increased MCE activity in malathion resistant strains. We then present a model in which the malathion carboxylesterase activity of the E3-Leu251 enzyme is enhanced in vivo by its OP hydrolase activity. The latter activity enables it to reactivate after phosphorylation by malaoxon, the activated form of malathion, accounting for the exceptionally high level of resistance to malathion. We conclude that the two types of resistance can be explained by kinetic changes caused by the two allelic substitutions in the E3 enzyme.
Resistance to organophosphorus insecticides (OPs) in the sheep blowfly, Lucilia cuprina, is associated with a non-staining phenotype of the carboxylesterase isozyme, E3 (E.C. 3.1.1.1). Here, we show that a member of α-esterase multigene family, LcαE7, encodes E3. An LcαE7 cDNA has been isolated from an OP-susceptible strain and expressed in a baculovirus. The expressed product is the same as E3 in its electrophoretic mobility and preference for α-over β-naphthyl acetate as substrate. Its preference (kcat/Km) for a range of carboxylester substrates is α-naphthyl butyrate>α-naphthyl propionate>α-naphthyl acetate>methylthiobutyrate> p-nitrophenyl acetate. The enzyme is potently inhibited by OPs (ki [paraoxon] = 6.3 ± 1.4 × 107 /M/min, ki [chlorfenvinphos] = 5.9 ± 0.6 × 107/M/min) and exhibits a high turnover of methylthiobutyrate (1009/s), consistent with its proposed homology to the ali-esterase that is thought to mutate to confer OP resistance in Musca domestica. E3 shares 64% amino acid identity with its Drosophila melanogaster homologue, DmαE7, and is also closely related to other esterases involved in OP resistance such as the B1 esterase of Culex pipiens (38%) and E4 of Myzus persicae (30%).
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.
A photometric method for determining acetylcholinesterase activity of tissue extracts, homogenates, cell suspensions, etc., has been described. The enzyme activity is measured by following the increase of yellow color produced from thiocholine when it reacts with dithiobisnitrobenzoate ion. It is based on coupling of these reactions: The latter reaction is rapid and the assay is sensitive (i.e. a 10 μ1 sample of blood is adequate). The use of a recorder has been most helpful, but is not essential. The method has been used to study the enzyme in human erythrocytes and homogenates of rat brain, kidney, lungs, liver and muscle tissue. Kinetic constants determined by this system for erythrocyte eholinesterase are presented. The data obtained with acetylthiocholine as substrate are similar to those with acetylcholine.
Esterases in organophosphate susceptible and resistant houseflies were studied by means of sensitive Gomori method. In susceptible flies the esterase activity to α-naphthylacetate was found to be mainly due to the cholinesterase and an ali-esterase identical with that responsible for most of the activity to methylbutyrate. The latter enzyme is much less active to β-naphthylacetate than to α-naphthylacetate.The Km values of the cholinesterase for α- and β-naphthylacetate were 1·0 × 10−4 and 2·3 × 10−4 (M) respectively. The ali-esterase had a Km of approximately 10−4 (M) for α-naphthylacetate.The activity to α-naphthylacetate of the cholinesterase was strongly, that of the ali-esterase only weakly pH-dependent in the range from pH 6 to 8. Both enzymes were more active at higher pH.Eserine and diazoxon were used in inhibition experiments, acetylcholine and methylbutyrate in experiments on substrate competition. The Km value of the cholinesterase for acetylcholine was calculated as approximately 10−5 (M).The addition of heat-inactivated homogenate strongly enhanced the ali-esterase activity to α-naphthylacetate. It did so much more at low than at high concentrations of the active homogenate and thus caused the disappearance of the disproportionality initially observed between enzyme concentration and activity. This activation phenomenon was, to a lesser extent, also observed with β-naphthylacetate.The ali-esterase activity to α-naphthylacetate in homogenates of organophosphate resistant strains was only about 15 per cent of that found in homogenates of organophosphate susceptible strains. No significant differences between the activities in susceptible and resistant strains were found if β-naphthylacetate and indophenylacetate were used as substrates.Agar-gel electrophoresis of the supernatants obtained by high-speed centrifugation of homogenates proved the presence of about seven electrophoretically different esterases that occurred in more or less strain-specific patterns.
Eight esterases identified using native polyacrylamide gel electrophoresis were characterized in a susceptible and an organophosphate-resistant strain of Lucilia cuprina. Developmental profiles and tissue distributions were determined for all eight esterases and substrate and inhibitor specificities were also determined for six of them. Esterases E1 and E2 were isozymes of acetylcholinesterase, with E1 present throughout development and E2 largely confined to adults. Esterases E8, which was mainly confined to the neuromuscular and digestive tissues of early larvae, and E7, which was only recovered from 2- to 3-day-old adults, were not abundant enough for biochemical analysis. E4 and E13, classified as carboxylesterases, were only moderately sensitive to inhibition by organophosphates and found mainly in hemolymph. E3 and E9 were also identified as carboxylesterases but were highly sensitive to inhibition by organophosphates. They were the only enzymes found to differ between the two strains, both being detected only in the organophosphate-susceptible strain. In this strain they were both abundant in pupae, and E3 was also abundant in larvae and sexually mature adults, localized mainly in digestive tissues and the adult reproductive tract. The inhibitor, developmental, and tissue specificities of E3 all support genetic data reported previously that E3 is encoded by the major organophosphate resistance locus, ROP-1. While genetic data are not available for E9, its concentration in pupae suggests that it is unlikely to confer major gene resistance in feeding stages of L. cuprina.
In insects, acetylcholinesterase is essential for life and its inhibition by organophosphorus or carbamate insecticides is lethal. Extensive use of these insecticides to control insect pests has led to resistant enzymes, less inhibited than the wild type enzyme. Some mutations responsible for resistance have been identified. The resulting mutated proteins present new catalytic properties towards substrates and inhibitors. The presence of an altered enzyme has several consequences on the genetics of resistance and maintenance of resistant alleles in natural populations.
Carboxylesterases from different strains of Myzus persicae were examined to try to understand their contribution to insecticide resistance. Preliminary evidence that they are involved comes from the good correlation between the degree of resistance and the carboxylesterase and paraoxon-degrading activity in aphid homogenates. Furthermore the carboxylesterase associated with resistance could not be separated from the insecticide-degrading enzyme by electrophoresis or ion-exchange chromatography. Homogenates of resistant aphids hydrolysed paraoxon 60 times faster than did those of susceptible aphids, yet the purified enzymes from both sources had identical catalytic-centre activities towards this substrate and also towards naphth-1-yl acetate, the latter being hydrolysed by both 2x10(6) times faster than paraoxon. These observations provide evidence that the enzyme from both sources is identical, and that one enzyme hydrolyses both substrates. This was confirmed by relating the rate of paraoxon hydrolysis to the rate at which paraoxon-inhibited carboxylesterase re-activated. Both had the same first-order rate constant (0.01min(-1)), showing clearly that the hydrolysis of both substrates is brought about by the same enzyme. Its K(m) for naphth-1-yl acetate was 0.131mm, and for paraoxon 75pm. The latter very small value could not be measured directly, but was calculated from substrate-competition studies coupled with measurements of re-activation of the diethyl phosphorylated enzyme. Since the purified enzymes from resistant and susceptible aphids had the same catalytic-centre activity, the 60-fold difference between strains must be caused by different amounts of the same enzyme resulting from mutations of the regulator gene(s) rather than of the structural gene.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
Recapitulation des travaux portant sur le mecanisme de resistance des insectes face aux substances toxiques, base sur l'amplification des genes de resistance. Evaluation du mecanisme, de cette amplification, de l'origine et de la stabilite des genes impliques
1. Acetylcholinesterase from the heads of a strain of houseflies selected for resistance to the carbamate insecticide methomyl, and from a methomyl-resistant field strain was found to be less sensitive to inhibition by methomyl than that from a susceptible strain. 2. The enzyme from resistant insects was also more tolerant to malaoxon, dichlorvos and bomyl but not to azamethiphos. 3. The decrease in sensitivity to inhibition appeared to be due to an increase in affinity for substrate.
Organophosphate (OP) insecticide resistance in certain strains of Musca domestica is associated with reduction in the carboxylesterase activity of a particular esterase isozyme. This has been attributed to a 'mutant ali-esterase hypothesis', which invokes a structural mutation to an ali-esterase resulting in the loss of its carboxylesterase activity but acquisition of OP hydrolase activity. It has been shown that the mutation in Lucilia cuprina is a Gly137-->Asp substitution in the active site of an esterase encoded by the Lc alpha E7 gene (Newcomb, R.D., Campbell, P.M., Ollis, D.L., Cheah, E., Russell, R.J., Oakeshott, J.G., 1997. A single amino acid substitution converts a carboxylesterase to an organophosphate hydrolase and confers insecticide resistance on a blowfly. Proc. Natl. Acad. Sci. USA 94, 7464-7468). We now report the cloning and characterisation of the orthologous M. domestica Md alpha E7 gene, including the sequencing of cDNAs from the OP resistant Rutgers and OP susceptible sbo and WHO strains. The Md alpha E7 gene has the same intron structure as Lc alpha E7 and encodes a protein with 76% amino acid identity to Lc alpha E7. Comparisons between susceptible and resistance alleles show resistance in M. domestica is associated with the same Gly137-->Asp mutation as in L. cuprina. Bacterial expression of the Rutgers allele shows its product has OP hydrolase activity. The data indicate identical catalytic mechanisms have evolved in orthologous Md alpha E7 and Lc alpha E7 molecules to endow diazinon-type resistance on the two species of higher Diptera.
Acetylcholinesterase (AChE), encoded by the Ace gene, is the primary target of organophosphorous (OP) and carbamate insecticides. Ace mutations have been identified in OP resistants strains of Drosophila melanogaster. However, in the Australian sheep blowfly, Lucilia cuprina, resistance in field and laboratory generated strains is determined by point mutations in the Rop-1 gene, which encodes a carboxylesterase, E3. To investigate the apparent bias for the Rop-1/E3 mechanism in the evolution of OP resistance in L. cuprina, we have cloned the Ace gene from this species and characterized its product. Southern hybridization indicates the existence of a single Ace gene in L. cuprina. The amino acid sequence of L. cuprina AChE shares 85.3% identity with D. melanogaster and 92.4% with Musca domestica AChE. Five point mutations in Ace associated with reduced sensitivity to OP insecticides have been previously detected in resistant strains of D. melanogaster. These residues are identical in susceptible strains of D. melanogaster and L. cuprina, although different codons are used. Each of the amino acid substitutions that confer OP resistance in D. melanogaster could also occur in L. cuprina by a single non-synonymous substitution. These data suggest that the resistance mechanism used in L. cuprina is determined by factors other than codon bias. The same point mutations, singly and in combination, were introduced into the Ace gene of L. cuprina by site-directed mutagenesis and the resulting AChE enzymes expressed using a baculovirus system to characterise their kinetic properties and interactions with OP insecticides. The K(m) of wild type AChE for acetylthiocholine (ASCh) is 23.13 microM and the point mutations change the affinity to the substrate. The turnover number of Lucilia AChE for ASCh was estimated to be 1.27x10(3) min(-1), similar to Drosophila or housefly AChE. The single amino acid replacements reduce the affinities of the AChE for OPs and give up to 8.7-fold OP insensitivity, while combined mutations give up to 35-fold insensitivity. However, other published studies indicate these same mutations yield higher levels of OP insensitivity in D. melanogaster and A. aegypti. The inhibition data indicate that the wild type form of AChE of L. cuprina is 12.4-fold less sensitive to OP inhibition than the susceptible form of E3, suggesting that the carboxylesterases may have a role in the protection of AChE via a sequestration mechanism. This provides a possible explanation for the bias towards the evolution of resistance via the Rop-1/E3 mechanism in L. cuprina.
Equations based on the assumption of a reversible first step in the reaction between organophosphate inhibitors and esterases
are proposed for the bimolecular rate constant which now includes an affinity constant and a phosphorylation constant. The
treatment applies when the inhibition reaction follows first-order kinetics.
Tiny insects become big problem . Stored Grain Australia, Canberra
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The Biochemistry and Use of Pesticides. Structure, Metabolism, Mode of Action and Use in Crop Protection
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Hassall, K. A., 1990: The Biochemistry and Use of Pesticides. Structure, Metabolism, Mode of Action and Use in Crop Protection, 2nd edn. New York: MacMillan, 536.
The mechanism of malathion resistance in the blowfly Chrysomyia putoria
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Van Asperen, K.; Oppenoorth, F. J., 1960: The mechanism of malathion resistance in the blowfly Chrysomyia putoria. Entomol. Exp. Appl. 3, 68–83.
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