General Esterase Research Articles

Evaluation of co-nanoencapsulation process on the toxicity and biochemical metabolism of imidacloprid and lambda-cyhalothrin in Myzus persicae (Sulzer)

The aphid Myzus persicae (Sulzer) is one of the most economically important plant pests worldwide. Excessive use of chemical pesticides to control this pest affects its successful management. In this study, we used a novel technology to improve insecticide efficacy while reducing doses through the combination of insecticides. Therefore, the joint action of pyrethroid lambda-cyhalothrin (LAM) and neonicotinoid imidacloprid (IMI) was evaluated on the aphid M. persicae using leaf-dipping method. Co-nanoencapsulated formulation of these insecticides was prepared using phosphatidylcholine and chitosan as carrier and surface-coating material, respectively. The results of toxicological studies revealed a considerable synergistic effect at 2:1 ratio of technical grade IMI plus LAM and their co-encapsulated formulation. Nanoparticles produced were spherical with size of 67.56 nm. The LC50 values for the technical grade of IMI, LAM and their mixture were 12.65, 89.82 and 4.07 mg ai l−1; for co-encapsulated formulation, the value was 7.06 mg ai l−1. To evaluate the effect of co-nanoencapsulation process on detoxification enzymes, the activities of general esterases and glutathione S-transferases and the amount of cytochrome P450s were measured in M. persicae. The technical grade mixture of IMI and LAM caused pronounced changes in the levels of all tested enzymes compared with the control. In contrast, co-nanoencapsulation protected IMI and LAM from cytochrome P450s- and glutathione S-transferase-induced enzymatic degradation. Considering the toxicological and biochemical findings, co-nanoencapsulation can be valuable as an eco-friendly method for improving the efficacy of the insecticides while preserving the synergistic effects and protecting the active ingredients against detoxification enzymes.

High-Throughput, Fluorescence-Based Esterase Activity Assay for Assessing Polysorbate Degradation Risk during Biopharmaceutical Development.

Hydrolytic degradation of polysorbate during 2-8°C storage of monoclonal antibody drug products has been attributed to residual enzymes (e.g., esterases) from bioprocessing steps. Robust detection of esterase activity using sensitive, non-polysorbate surrogate substrates can provide an alternate method to assess polysorbate degradation risk, if the correlation between the esterase activity and polysorbate degradation is established. A general esterase activity assay was developed as a monitoring and characterization tool during bioprocess development of monoclonal antibodies. We report a fluorescence plate-based assay for quantifying esterase activity, utilizing 4-methylumbelliferyl caprylate (MU-C8) as the esterase substrate. The assay was first assessed for substrate, inhibitor and pH specificity using both model enzymes and purified protein samples. The assay was then extensively tested to understand sample matrix effects on activity rates. The use of this high-throughput method will allow for rapid characterization of protein samples in under three hours. The esterase activity correlated directly with polysorbate degradation and can provide valuable information on polysorbate degradation risk throughout drug development.

Insecticide resistance among field populations of Hyposidra talaca Walker (Geometridae: Lepidoptera) in tea plantations of Assam, India: detection through a biochemical approach

The black inch looper, Hyposidra talaca is a serious defoliator pest of tea and its management relies profoundly on application of insecticides. Here, we investigated the insecticide susceptibility in H. talaca collected from six districts of Assam to five insecticides: bifenthrin and deltamethrin (Pyrethroids), quinalphos (Organophosphates), emamectin benzoate (Avermectins) and flubendiamide (Diamides). Based on relative susceptibility values, all populations showed varied levels of susceptibility to the insecticides evaluated as compared to the susceptible population. Tinsukia and Dibrugarh populations showed high LC50s to most of the insecticides assessed. A moderate to high level of resistance was recorded for deltamethrin [Resistance coefficient (RC) = 19.03–31.75; Resistance ratio (RR) = 8.23–38.90]. Bifenthrin showed moderate level of resistance [RC = 11.80-23.79; RR = 16.52–25.52] while quinalphos demonstrated low-resistance level [RC = 4.35–6.06; RR = 3.22–4.41]. Emamectin benzoate and flubendiamide were effective against H. talaca in all tested populations, except for Tinsukia and Dibrugarh populations where low levels of resistance were recorded. Detoxification enzyme assays indicate that glutathione S-transferases (GST), general esterase (GE) and cytochrome P450 (CYP) activities were high in all field populations as compared to susceptible ones. GE and CYP had significant variation among different population and registered positive correlation with the LC50s of deltamethrin, bifenthrin and quinalphos. Synergistic assays reveal that the use of synergists’ piperonyl butoxide, S.S.S.-Tributylphosphorotrithioate and ethacrynic acid in combination with deltamethrin and quinalphos re-establishes the toxicity of these insecticides. The findings show that detoxification enzymes play important role in the development of resistance to insecticides in H. talaca and will be useful in designing resistance management programmes against the pest.

Can Organophosphates and Carbamates Cause Synergisms by Inhibiting Esterases Responsible for Biotransformation of Pyrethroids?

Hydrolysis catalyzed by general esterases (GEs) is the most efficient route for hydrolyzation of pyrethroid insecticides. Organophosphate (OP) and carbamate (CB) insecticides are known to inhibit GEs in addition to acetylcholinesterase (AChE), which is their main target. We hypothesize that synergies can be induced by OPs and CBs when mixed with pyrethroids, due to their inhibition of GE-dependent detoxification of pyrethroids. To test this hypothesis, we conducted mixture toxicity experiments with Daphnia magna using α-cypermethrin (α-cyp) in combination with the noninsecticidal OP tetraisopropyl pyrophosphoramide (iso-OMPA) and five AChE inhibitors diazinon, chlorpyrifos, chlorfenviphos, parathion, and aldicarb. In addition, the in vivo GE activity inhibition was measured for all compounds. Up to 10-fold synergy was found between α-cyp and iso-OMPA, and the degree of synergy correlated linearly with the inhibition of the GE activity. No synergy, however, was found in any of the insecticide mixtures nor was the GE activity inhibited within the nonlethal concentration range tested. It was concluded that the effect of the insecticides on AChE occurred at lower concentrations than their effect on GEs, making the daphnids become immobilized before any synergistic effects on mortality could be observed. The implications of the findings are discussed from a risk assessment perspective.

Monophosphoramide derivatives: synthesis and crystal structure, theoretical and experimental studies of their biological effects.

Synthesizing new chemical compounds and studying their biological applications have been important issues in scientific research. In this investigation, we synthesized and characterized ten new N-acetyl phosphoramidate compounds and explored the crystal structure of three others. Furthermore, not only were some kinetic inhibition parameters measured, like IC50, Ki, kp, KD for 7 compounds on human acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), but also their hydrophobic parameter was determined by shake-flask technique. All compounds (number 1-10) were investigated for anti-bacterial activity against three Gram-positive and three Gram-negative bacteria, while chloramphenicol was used as a standard antibiotic. In order to find new insecticide, toxicities of 13 acephate (Ace)-derived compounds (number 20-32) were bioassayed on third larval instar of elm leaf beetle and Xanthogaleruca luteola. Additionally, screening in vivo tests revealed that two compounds had had the greatest insecticidal potential in comparison with others. It means these ones inhibited AChE (with mixed mechanisms) and general esterase more than the rest. According to ChE-QSAR models, the inhibitory potency for enzyme and bacteria is directly influenced by the electronic parameters versus structural descriptors. AChE-QSPR model of fluorescence assay indicated that the inhibitory power of AChE is primarily influenced by a set of electronic factors with the priority of: EHB > PL > δ(31P) versus structural descriptor (SA and Mv). Synthesizing new chemical compounds and studying their biological applications have been important issues in scientific research. Toxicities of 13 acephate (Ace)-derived compounds (number 20-32) were bioassayed on third larval instar of elm leaf beetle and Xanthogaleruca luteola. Insect-QSAR equations of these compounds, based on MLR and PCA, showed that non-descriptor net charge nitrogen atom (which was affected by the polarization of N-H group) had the greatest effect on insecticidal potential.

Stage specific differential expression of three detoxifying enzymes of larvae of tea defoliator, Hyposidra talaca Walker (Geometridae: Lepidoptera) and its bearing on their insecticide tolerance status

The black inch looper of Hyposidra talaca is one of the important defoliating pests of tea in north-eastern India which is capable of completing 6–8 generations in a year without any diapause. The looper stages of H. talaca passes through five larval instars causing considerable damage to the tea crop. The effective control of H. talaca greatly relies on application of synthetic insecticides. In the present study, we determined the relative toxicity of two selected insecticides viz., deltamethrin and quinalphos along with differential expression of the three detoxifying enzymes (general esterase, glutathione S-transferase, and cytochrome-P450-monooxygenase) in various larval stages of H. talaca populations collected from the tea ecosystem. In general, both insecticides were most active against first and second stage larvae but their toxicity levels decreased with the advancement of life stages. The expression of the three detoxifying enzymes in all five larval stages varied greatly which increased with each successive stage suggesting that these enzymes are serving as defensive mechanism for the insects to insecticidal stress. These results show that sensitivity of insecticides was directly related to the stage of the insect and suggests that chemical treatment at early stages of the insect is more effective than at later stages. Results of the present work reveal the mechanisms underlying in the adaptation of H. talaca to insecticides which would be of considerable significance for the development of effective pest management strategies.

Fumigant toxicity of three Satureja species on tomato leafminers, Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae)

The tomato leafminer, Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae), is one of the major insect pests of solanaceous crops especially tomatoes. As plants contain secondary metabolites with protective properties against unwanted agents, they can be used as insecticides after extraction from plants as essential oils (EOs). In this study, essential oils of three savory species, including Satureja khuzestanica, S. bachtiarica, and S. rechingeri were extracted. Analyzing the chemical compositions via gas chromatography–mass spectrometry (GC/MS) revealed that geraniol was the main component of all essential oils. The volatile activity of these three savory species on the fourth instar larvae of T. absoluta during 24 h, indicated that LC50 values of S. khuzestanica, S. bachtiarica, and S. rechingeri oils were 17.51, 25.03, and 34.33 μgL−1(air), respectively. Enzyme assays in vivo using alpha-naphthyl acetate (α-NA) as a substrate, showed that in the fourth instar larvae treated with 10 percent lethal concentration (LC10), general esterases of these larvae increased significantly in all treatments (p < .05). Polyacrylamide gel electrophoresis with a solution mixture of both substrates (α-and ß-NA) clarified three bands of esterases. However, this method failed to show any sublethal effects of the oils (LC10 value) on the larvae esterase bands. Considering the potential toxicity of these three species of Satureja essential oils to the tomato leafminers due to existence of several secondary metabolites, we can expect that new bio-pesticides can be developed from these promising plants in near future.

Effects of emamectin benzoate combined with acetamiprid, eforia and hexaflumuron against Tuta absoluta (Lep.: Gelechiidae)

Tomato leafminer, Tuta absoluta Meyrick (Lepidoptera: Gelechiidae) is one of the most destructive pests of tomato in many parts of the world including Iran. The toxicity of emamectin benzoate alone and combined with acetamiprid, eforia (thiamethoxam + lambda-cyhalotrin) and hexaflumuron was evaluated against 2nd instar larvae of Tuta absoluta (Meyrick) under 25 ± 1 °C, the relative humidity of 75 ± 5% and a photoperiod of 16:8 (L:D). Also the mixtures of tested insecticides with emamectin benzoate at LC15:LC15 ratio were evaluated on the activity of general esterase enzyme and total protein content of 2nd instar larvae. After 96 hour, emamectin benzoate had the highest toxicity (LC50 = 0.52 mg A.I./L), followed by acetamiprid (LC50 = 56.39 mg A.I./L) and eforia (LC50 = 312.01 mg A.I./L). Hexaflumuron showed no toxicity against larvae. The combination of emamectin benzoate with acetamiprid at LC50:LC50 ratio produced synergistic effects and all of the other ratios showed additive effects. The emamectin benzoate combined with either eforia or hexaflumuron at all of the ratios produced additive and antagonistic effects, respectively. Esterase activity of larvae increased when emamectin benzoate was mixed with either acetamiprid or eforia, but no significant differences were seen between emamectin benzoate alone and its mixture with hexaflumuron. The combination of emamectin benzoate with tested insecticides significantly reduced the total protein content of larvae. According to the results of this study, the mixtures of acetamiprid and eforia with emamectin benzoate showed higher negative impacts against 2nd instar larvae than emamectin benzoate alone

Standardization of tick specific biochemical tools for estimation of esterases, monooxygenases and glutathione S-transferase for characterization of acaricide resistance

Protocols to determine metabolic resistance in ticks were mainly derived from reports published using mosquitoes and agriculturally important insects without prior standardization. In the present study, biochemical assays were standardized to quantify acaricide metabolizing enzymes in tick homogenates. Three variables viz., age, number of larvae and reaction time were optimized using reference susceptible IVRI-I and deltamethrin resistant IVRI-IV (Resistance Factor = 194) tick strains. The optimum conditions for estimation of general esterases were 10–15 day old 40 larvae with 15 mins reaction time, 15–20 day old 40 larvae with 20 mins reaction time for Glutathione S- transferase, while 10–15 day old 80 larvae with 5 mins reaction time for monooxygenase. The standardized protocols were further validated in multi acaricide resistant strain (IVRI-V) and in nine field isolates having variable resistant factors to different acaricides. In all the nine heterogeneous field isolates, a significant correlation (p < .05) between resistance to synthetic pyrethroids and over-expression of esterases and monooxygenase was noticed. Similarly, esterases and GST activities were significantly correlated with resistance to organophosphates. The details of the assay protocol are explained for adoption in different laboratories.

Detection and biochemical characterization of acaricide resistance in field populations of tea red spider mite, Oligonychus coffeae (Acari: Tetranychidae), in Assam tea plantation of India

ABSTRACTThe red spider mite, Oligonychus coffeae, (RSM) is one of the most damaging pests of tea in most tea-producing countries. The use of synthetic acaricide, the main strategy for controlling RSM, has increased year by year. In this study, eight field populations of RSM were evaluated for resistance against five different acaricides (ethion, dicofol, propargite, fenazaquin, and fenpropathrin) using the leaf-dip method. The results showed that almost all the tested field populations had developed a high to very high level of resistance to ethion and dicofol. The highest level of resistance for RSM was found to ethion and dicofol with a resistance ratio of 134.27 and 65.38, respectively in the Cachar population as compared to the laboratory susceptible population. Propargite and fenazaquin were very effective against RSM in all tested populations, whereas a minor level of tolerance was evident against fenpropathrin at the recommended dose. Biochemical assays using detoxifying enzymes indicated that general esterase, glutathione-S- transferase, and cytochrome P450 monooxygenase activities were higher in all conventional field populations than in the susceptible population and also had a significantly positive correlation with the LC50 values for ethion and dicofol. Synergism assays were therefore performed using ethion and dicofol in combination with common synergists like piperonylbutoxide (PBO), S.S.S.-Tributylphosphorotrithioate (DEF), and ethacrynic acid (EA). Results showed that mixing of PBO with ethion ensured about 36.73 times higher toxicity against RSM. DEF was moderately effective in revamping the effect of ethion whereas only EA was able to reduce the tolerance of RSM population to dicofol up to 11.71 times. The present studies infer that general esterase and cytochrome P450 monooxygenase may play a major role in imparting resistance to ethion whereas only glutathione-S-transferase plays an important role in dicofol resistance in RSM. The present study indicates the need for adopting integrated resistance management programmes to effectively control RSM in the tea plantations of Assam.

Bacteria-mediated modification of insecticide toxicity in the yellow fever mosquito, Aedes aegypti

The incidence of mosquito-borne disease poses a significant threat to human and animal health throughout the world, with effective chemical control interventions limited by widespread insecticide resistance. Recent evidence suggests that gut bacteria of mosquitoes, known to be essential in nutritional homeostasis and pathogen defense, may also play a significant role in facilitating insecticide resistance. This study investigated the extent to which bacteria contribute to the general esterase and cytochrome P450 monooxygenase (P450)-mediated detoxification of the insecticides propoxur and naled, as well as the insecticidal activity of these chemistries to the yellow fever mosquito, Aedes aegypti. Experiments conducted using insecticide synergists that reduce general esterase and P450 activity demonstrate a role for both groups of enzymes in the metabolic detoxification of propoxur and naled. Furthermore, reduction of bacteria in mosquito larvae using broad-spectrum antibiotics was found to decrease the metabolic detoxification of propoxur and naled, suggesting that the bacteria themselves may be contributing to the in vivo metabolic detoxification of these insecticides. This was supported by in vitro assays using culturable gut bacteria isolated from mosquito larvae which demonstrated that the bacteria were capable of reducing insecticide toxicity. More work is needed, however, to fully elucidate the contribution of bacteria in Ae. aegypti larvae to the metabolic detoxification of insecticides.

Status of insecticide resistance and its biochemical and molecular mechanisms in Anopheles stephensi (Diptera: Culicidae) from Afghanistan

BackgroundInsecticide resistance of Anopheles stephensi, the main malaria vector in eastern Afghanistan, has been reported previously. This study describes the biochemical and molecular mechanisms of resistance to facilitate effective vector control and insecticide resistance management.MethodsMosquito larvae were collected from the provinces of Kunar, Laghman and Nangarhar from 2014 to 2017. The susceptibility of the reared 3–4 days old adults was tested with deltamethrin 0.05%, bendiocarb 0.1%, malathion 5%, permethrin 0.75% and DDT 4%. Cytochrome P450 content and general esterase, glutathione S-transferase (GST) and acetylcholinesterase (AChE) activities were measured in the three field populations and the results were compared with those of the laboratory susceptible An. stephensi Beech strain. Two separate allele-specific PCR assays were used to identify L1014, L1014F and L1014S mutations in the voltage gated sodium channel gene of An. stephensi. Probit analysis, ANOVA and Hardy–Weinberg equilibrium were used to analyse bioassay, biochemical assay and gene frequency data respectively.ResultsThe population of An. stephensi from Kunar was susceptible to bendiocarb, apart from this, all populations were resistant to all the other insecticides tested. The differences between all values for cytochrome P450s, general esterases, GSTs and AChE inhibition rates in the Kunar, Laghman and Nangarhar populations were statistically significant when compared to the Beech strain, excluding GST activities between Kunar and Beech due to the high standard deviation in Kunar. The three different sodium channel alleles [L1014 (wild type), L1014F (kdr west) and L1014S (kdr east)] were all segregated in the Afghan populations. The frequencies of kdr east mutation were 22.9%, 32.7% and 35% in Kunar, Laghman and Nangarhar populations respectively. Kdr west was at the lowest frequency of 4.44%.ConclusionsResistance to different groups of insecticides in the field populations of An. stephensi from Kunar, Laghman and Nangarhar Provinces of Afghanistan is caused by a range of metabolic and site insensitivity mechanisms, including esterases, cytochrome P450s and GSTs combined with AChE and sodium channel target site insensitivity. The intensity and frequency of these mechanisms are increasing in these populations, calling for urgent reorientation of vector control programmes and implementation of insecticide resistance management strategies.

Quantification of the activity of detoxifying enzymes in terrestrial invertebrates: Optimization, evaluation and use of in vitro and ex vivo methods

Abstract The negative effects of pesticides on beneficial terrestrial invertebrates have received much attention, and in this context quantification of the activity of metabolic enzymes provides an important tool. Unfortunately, current methodologies are limited in their use. In this study, the grain beetle, Tenebrio molitor was used to illustrate why in vitro methods are ineffective to quantify metabolic enzymes in some insects. It was further used to optimize an ex vivo assay that can replace in vitro methods when these are ineffective. The ex vivo method can quantify metabolic enzymatic activities of cytochrome P450 mixed function oxidases (CYPs), general esterases (EST) and glutathione S‐transferases (GST). In detail we show, that T. molitor produces inhibitory and/or degrading agents, which are released during tissue homogenization. As these agents inhibit the in vitro CYP activity of rat liver microsomes we suggest, that they are also responsible for the lack of in vitro CYP activity of T. molitor. We tested the effectiveness of broad‐spectrum protease inhibitors as protective agents and found, that they were only effective in concentrations that also interfered directly with the activity of CYP enzymes. As an alternative to in vitro measurements, we optimized an ex vivo method for quantification of CYP, EST and GST on individual gut systems of T. molitor. Our ex vivo method advances previous ex vivo methods, by allowing quantification of three major enzyme families, all in a single individual, and by exploiting real‐time kinetic measurements of the enzymatically produced product. Subsequently, our method was successfully tested for its use on three different life stages: larvae, pupae, and adult insects from T. molitor and Apis mellifera. The method shows promise for expansion to other species and potentially other enzyme families and/or substrates.

“Armed to the teeth”: The multiple ways to survive insecticidal and predatory challenges in Aedes aegypti larvae

Environmental pollutants, such as insecticides, can alter the equilibrium of aquatic ecosystems, particularly those closely located to human occupations. The use of such anthropogenic compounds frequently results in the selection of resistant individuals. However, how the underlying insecticide resistance mechanisms interplay with the abilities of the resistant individuals to cope with other environmental challenges (e.g., predators) has not received adequate attention. Here, we evaluated whether resistance to pyrethroid insecticides in larvae of the yellow fever mosquito, Aedes aegypti (Diptera: Culicidae), would affect their abilities to survive other environmental challenges. We assessed the susceptibilities of the pyrethroid-resistant larvae to other insecticides (i.e., the oxadiazine indoxacarb and juvenile hormone mimic pyriproxyfen) and determined the activities of potential detoxification enzymes. Finally, we also recorded potential alterations in larva swimming behavior in the presence of predators, such as the water bug Belostoma anurum (Hemiptera: Belostomatidae). Our results revealed that high pyrethroid resistance was associated with moderate resistance to the other two insecticides. Furthermore, this multiple resistance was associated with higher detoxification activity by glutathione-S-transferases and general esterases. Interestingly, in comparison with insecticide-susceptible larvae, the pyrethroid-resistant larvae not only swam for longer periods and distances, but also took longer to be captured by B. anurum nymphs. Collectively, our findings revealed increased abilities to survive natural environmental challenges (e.g., predatory attacks) in mosquito larvae that express physiological and behavioral changes associated with multiple resistance to insecticides.

F331H mutation and reduced mRNA in type-1 acetylcholinesterase is associated with carbofuran resistance development in the small brown planthopper (Laodelphax striatellus Fallén)

Molecular diagnostic markers are necessary for establishing high-throughput screening systems to support insecticide-resistant population management. Here, we identified single amino acid substitution mutations related to carbamate resistance in Laodelphax striatellus Fallén type-1 acetylcholinesterase (LsAChE1) using carbofuran-selected strains. The phenotypic resistance profiles of the final selection strain (SEL9) compared to the susceptible strain revealed a 14-fold higher resistance ratio based on topical application, 1.2-fold higher general esterase activity, and 4.3-fold higher acetylcholinesterase insensitivity based on the 50% inhibitory concentration (I50), suggesting that insensitivity of the target site could occur as a resistance factor. Comparison of the nucleotide sequences of Lsace1 of five strains (SUS, SEL0, SEL3, SEL6, and SEL9) revealed two amino acid substitutions (F330Y and F331H). To understand the roles of these mutations, we determined the allele frequency of both point mutations in the selected strains using quantitative sequencing methods. In addition, several quantitative genotypic traits (e.g., gene copy numbers and transcript levels of Lsace1, Lsace2, and LS.CarE1) were assessed. A correlation analysis of genotypic and phenotypic traits revealed strong correlations between resistance level and I50 with F331H allele frequency. Interestingly, the F331H mutation was negatively correlated with transcript levels of Lsace1, suggesting that selection pressure might result in a reduction of the target gene. Overall, the F331H mutation and reduced mRNA are important factors in the development of carbamate resistance. Furthermore, the point mutation can be used to monitor rapid carbofuran resistance in conjunction with molecular diagnostic methods such as quantitative sequencing.

Detection and biochemical characterization of insecticide resistance in field populations of Asian citrus psyllid in Guangdong of China

The Asian citrus psyllid, Diaphorina citri Kuwayama, is one of the most damaging pests of citrus-producing regions throughout the world. The use of insecticides is the main strategy for controlling psyllid and has increased year by year. In this study, four field populations of D. citri were evaluated for resistance to nine different insecticides using the leaf-dip method. The results showed that the highest level of resistance for D. citri was found in imidacloprid with a resistance ratio of 15.12 in the Zengcheng population compared with the laboratory susceptible population. This was followed by chlorpyriphos (6.47), dinotefuran (6.16), thiamethoxam (6.04), lambda-cyhalothrin (4.78), and bifenthrin (4.16). Piperonyl butoxide (PBO) and triphenyl phosphate (TPP) showed significant synergism on imidacloprid effects in the Zengcheng population (3.84- and 2.46-fold, respectively). Nevertheless, diethyl maleate (DEM) had no synergism on imidacloprid. Biochemical enzyme assays suggested that general esterase, glutathione S-transferase and cytochrome P450 monooxygenase activities were higher in the field-collected populations than in the laboratory susceptible population. However, glutathione S-transferase may play a minor role in the resistance of adult D. citri to insecticides. At the molecular level, resistance of D. citri to imidacloprid is mainly related to the increased expression of CYP4C68 and CYP4G70 (>5-fold).

Resistance to Permethrin, β-cyfluthrin, and Diazinon in Florida Horn Fly Populations.

Horn flies, Haematobia irritans, a major cattle pest in the USA, cause substantial economic losses and current control methods rely heavily on insecticides. Three horn fly populations were evaluated for insecticide susceptibility to permethrin, β-cyfluthrin, and diazinon. Susceptibility was variable by population, with the greatest resistance exhibited by a 66-fold resistance ratio (RR) to permethrin and >14-fold RR to diazinon. Mechanisms of resistance were determined using molecular techniques and enzymatic assays. The knockdown resistance (kdr) genotype (L150F) associated with pyrethroid resistance, and a G262A mutation in acetylcholinesterase, previously associated with organophosphate resistance, were found in all field populations evaluated. Insensitivity of diazoxon at the acetylcholinesterase (AChE) target site was significantly different in horn flies from one of the field sites. For metabolic detoxifying enzymes, cytochrome P450 nor general esterases showed a significant difference between field strains and a laboratory susceptible strain. Pyrethroid resistance was likely due to the presence of the L150F mutation in the population. In vitro studies targeting the AChE enzyme did not support the notion that the G262A mutation was the sole cause of resistance to organophosphates, and, therefore, the exact resistance mechanism to diazinon was not able to be confirmed.

Susceptibility to acaricides and detoxifying enzyme activities in the red spider mite, Oligonychus coffeae Nietner (Acari: Tetranychidae)

Susceptibility of red spider mite, Oligonychus coffeae Nietner (Acari: Tetranychidae), collected from conventionally-managed (synthetic acaricide usage) versus an organicallymanaged (no acaricide usage) tea plantations in Assam, India, to five synthetic acaricides was determined in laboratory bioassays. Activity of three principal detoxifying enzymes of these mite populations was also assayed. The median lethal concentrations (LC50) of ethion, dicofol, propargite, fenpropathrin, and fenazaquin were 1049.75, 599.21, 232.03, 11.44, and 6.75 ppm, respectively. Field rates of these acaricides were compared with 95% lethal concentration (LC95 in ppm) values, and a decrease in the susceptibility of the test population to ethion, propargite, dicofol and fenpropathrin was observed. There was no change for fenazaquin which was effective at lower doses than the recommended dose. Of all the acaricides tested, fenazaquin was the most toxic and ethion was the least toxic. General esterases (GEs), glutathione- S-transferase (GST), and cytochrome P450 monooxygenases exhibited a higher activity in mite population from the conventionallymanaged tea plantation as compared with the activity in mites from the organicallymanaged tea plantation. These findings may be helpful in the selection of acaricides and in developing resistance management strategies for an effective management program for this major tea pest.

Sex-based variation in insecticide susceptibility and tolerance related biochemical parameters in tea mosquito bug Helopeltis theivora

Helopeltis theivora, one of the major pests of tea from North east India, reported to acquire tolerance against commonly used synthetic pesticides. Study on relative toxicity of both sexes of H. theivora along with their detoxifying enzyme levels were carried out to assess developemnt of sex-based tolerance of pest against the pesticides. The present study showed that female develop more tolerance among H. theivora population than male. Relative toxicity tests showed that LC50 was higher in female than male H. theivora. This result reflects in general, sex level difference in tolerance status of the pest along with corresponding changes in the quantities of detoxifying enzymes. Female of H. theivora showed significantly higher general estearse and glutathione-S-transferase activities than that of male; however there was no significant difference in cytochrome P450 activity. The total body lipid content was also found to be higher in female than male, which indicated female attribute to be more tolerant than male. Body weight of female was significantly higher than that of male, which showed positive correlation with general esterase and glutathione-S-transferase. Hence, on application of recommended dose of pesticide, chance of survivability of female is better in field, leading to addition of more tolerant forms.

Insecticide toxicity associated with detoxification enzymes and genes related to transcription of cuticular melanization among color morphs of Asian citrus psyllid.

The Asian citrus psyllid (Diaphorina citri Kuwayama) is known to exhibit abdominal color polymorphisms. In the current study, susceptibility to four insecticides was compared among orange/yellow, blue/green and gray/brown color morphs of field collected D. citri. The LD50 values and 95% fiducial limits were quantified for each insecticide and color morph combination and ranged between 0.10 ng/μL (0.06-0.10) and 6.16 ng/μL (3.30-12.50). Second, we measured the detoxification enzyme activity levels of orange/yellow, blue/green and gray/brown color morphs for cytochrome P450, glutathione S-transferase, and general esterase. The mean P450 activity (equivalent units) was significantly lower in gray/brown (0.152 ± 0.006) and blue/green morphs (0.149 ± 0.005) than in the orange/yellow morphs (0.179 ± 0.008). GST activity (μmol/min/mg protein) was significantly lower in the orange/yellow morph (299.70 ±1.24) than gray/brown (350.86 ± 1.19) and blue/green (412.25 ± 1.37) morphs. The mean EST activity (μmol/min/mg protein) was significantly higher in blue/green (416.72 ± 5.12) and gray/brown morphs (362.19 ± 4.69) than in the orange/yellow morphs (282.56 ± 2.93). Additionally, we analyzed the relative expression of assortment genes involved in cuticular melanization and basal immunity. The transcripts of Dopa Decarboxylase and Tyrosine Hydroxylase were expressed higher in blue/green and gray/brown than orange/yellow morphs. The transcription results paralleled the susceptibility of D. citri to organophosphate, neonicotinoid and pyrethroid insecticides. GST and EST activity may also be correlated with low levels of insecticide susceptibility. Cuticular melanization could be a factor for the development of resistance to insecticides among different color morphs.