Mixed Function Oxidase Activity Research Articles

The Comparative Toxicity, Biochemical and Physiological Impacts of Chlorantraniliprole and Indoxacarb on Mamestra brassicae (Lepidoptera: Noctuidae).

The cabbage moth, Mamestra brassicae, is a polyphagous pest that attacks several crops. Here, the sublethal and lethal effects of chlorantraniliprole and indoxacarb were investigated on the developmental stages, detoxification enzymes, reproductive activity, calling behavior, peripheral physiology, and pheromone titer of M. brasssicae. Methods: To assess pesticide effects, the second instar larvae were maintained for 24 h on a semi-artificial diet containing insecticides at their LC10, LC30, and LC50 concentrations. M. brassicae was more susceptible to chlorantraniliprole (LC50 = 0.35 mg/L) than indoxacarb (LC50 = 1.71 mg/L). A significantly increased developmental time was observed with both insecticides at all tested concentrations but decreases in pupation rate, pupal weight, and emergence were limited to the LC50 concentration. Reductions in both the total number of eggs laid per female and the egg viability were observed with both insecticides at their LC30 and LC50 concentrations. Both female calling activity and the sex pheromone (Z11-hexadecenyl acetate and hexadecenyl acetate) titer were significantly reduced by chlorantraniliprole in LC50 concentration. Antennal responses of female antennae to benzaldehyde and 3-octanone were significantly weaker than controls after exposure to the indoxocarb LC50 concentration. Significant reductions in the enzymatic activity of glutathione S-transferases, mixed-function oxidases, and carboxylesterases were observed in response to both insecticides.

Insecticide resistance levels and associated mechanisms in three Aedes aegypti populations from Venezuela.

The massive use of insecticides in public health has exerted selective pressure resulting in the development of resistance in Aedes aegypti to different insecticides in Venezuela. Between 2010 and 2020, the only insecticides available for vector control were the organophosphates (Ops) fenitrothion and temephos which were focally applied. To determine the state of insecticide resistance and to identify the possible biochemical and molecular mechanisms involved in three populations of Ae. aegypti from Venezuela. CDC bottle bioassays were conducted on Ae. aegypti collected between October 2019 and February 2020 in two hyperendemic localities for dengue in Aragua State and in a malaria endemic area in Bolívar State. Insecticide resistance mechanisms were studied using biochemical assays and polymerase chain reaction (PCR) to detect kdr mutations. Bioassays showed contrasting results among populations; Las Brisas was resistant to malathion, permethrin and deltamethrin, Urbanización 19 de Abril was resistant to permethrin and Nacupay to malathion. All populations showed significantly higher activity of mixed function oxidases and glutathione-S-transferases (GSTs) in comparison with the susceptible strain. The kdr mutations V410L, F1534C, and V1016I were detected in all populations, with F1534C at higher frequencies. Insecticide resistance persists in three Ae. aegypti populations from Venezuela even in the relative absence of insecticide application.

Detoxification enzyme activity, reproductive and developmental fitness of abamectin-resistant Bryobia praetiosa (Acari:Tetranychidae)

Abamectin is an important and environmentally friendly antibiotic pesticide, and its resistance has become an area of intense research. In this study, we employed laboratory bioassays, biochemical analyses, and life history analyses to systematically evaluate abamectin resistance, the underlying biochemical mechanisms of resistance, and the reproductive and developmental characteristics of the arthropod pest Bryobia praetiosa. An abamectin-resistant B. praetiosa strain (RR) was obtained by intensive selection of an abamectin-susceptible strain (SS) using abamectin exposure in the laboratory for 24 generations, yielding a resistance index (RI) of 30.11. Comparison of detoxification enzymes suggested that the specific activities of carboxylesterases (CarEs), glutathione S-transferases (GSTs), and mixed-function oxidases (MFOs) in the RR strain were higher than those of the SS strain by 1.13-, 1.75-, and 4.02-fold, respectively. GST and MFOS protein concentrations were also significantly different between RR and SS strains. Further analysis of life history parameters demonstrated that the developmental duration and lifespan of RR strain in addition to egg numbers were markedly decreased relative to SS strain. The net reproductive rate (R0), finite rate of increase (λ), and population doubling time (Dt) of RR strain were significantly lower than those of SS strain. The results indicated that the RR strain was more disadvantageous in developmental and reproductive characteristics than the SS strain. In addition, the relative fitness value of the resistant strain is about half (Rf = 0.49) that of the susceptible strain. In conclusion, increased abamectin resistance in B. praetiosa was associated with a rapid increase in MFOs activity, while GSTs also played a role in abamectin resistance development and RR strain exhibited reproductive disadvantages.

Magnitude of Resistance and Metabolism of Acaricides in Two-spotted Spider Mite, Tetranychus urticae Koch on Vegetables in Southern Districts of Tamil Nadu

Background: Two-spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae) is a destructive, polyphagous and cosmopolitan pest infesting a wide range of vegetables. Farmers frequently use acaricides in the vegetable ecosystem for mite management leading to increased probability for the development of resistance against acaricides in mite species. Literature about resistance in T. urticae to novel acaricides is scanty from this region. Hence, this study was aimed to evaluate the magnitude of acaricide resistance in T. urticae populations on vegetable crops from Southern districts of Tamil Nadu against seven acaricides. The role of detoxification enzymes in conferring bio-chemical resistance in the mite species was also studied. Methods: The laboratory experiments were conducted at completely randomized design (CRD) during 2019-2021 in central instrumentation laboratory, Department of Agricultural Entomology, Agricultural College and Research Institute, Tamil Nadu Agricultural University, Madurai. The level of resistance in T. urticae populations to fenazaquin, propargite, spiromesifen, buprofezin, fenpropathrin, diafenthiuron and chlorfenapyr was evaluated following the leaf dip bioassay technique. The activity of the detoxifying enzymes viz., Glutathione S Transferase (GST), Mixed Function Oxidase (MFO) and Carboxylesterase (CarE) were estimated. Result: Fenazaquin was highly toxic to T. urticae, which recorded a low level of resistance (2.00 to 8.62-fold). T. urticae exhibited a low to moderate level of resistance to fenpropathrin (1.86 to 37.28-fold), moderate to a high level of resistance to diafenthiuron (15.81 to 50.53-fold), high level of resistance to propargite (45.16 to 65.10-fold) and chlorfenapyr (54.67 to 100.14-fold) and extremely high level of resistance to spiromesifen (193.04 to 452.61-fold) and buprofezin (377.97 to 514.44-fold). Among all the test populations, the highly resistant DAmRg population exhibited the highest specific activity of the detoxifying enzymes, viz., GST (26.98 nmoles ml-1 min-1 mg of protein-1), MFO (5.07 nmoles ml-1 min-1 mg of protein-1) and CarE (827.90 nmoles ml-1 min-1 mg of protein-1). The CarE activity was found to be positively correlated with highly resistant acaricide, propargite (r = 0.995) and MFO activity with buprofezin having an extremely high level of resistance (r = 0.997). This preliminary knowledge would be instrumental in framing the acaricide resistant management (ARM) strategies.

Assessment of Sex-Specific Toxicity and Physiological Responses to Thymol in a Common Bean Pest Acanthoscelides obtectus Say.

Acanthoscelides obtectus Say (Coleoptera: Chrysomelidae: Bruchinae), is one of the most important pests of the common bean Phaseolus vulgaris L. Without appropriate management it may cause significant seed loss in storages. In search for means of environmentally safe and effective protection of beans we assessed biological activity of thymol, an oxygenated monoterpene present in essential oils of many aromatic plants. We studied contact toxicity of thymol on bean seeds and its effects on adult longevity and emergence in F1 generation. Furthermore, we determined acetylcholinesterase (AChE), superoxide dismutase (SOD), catalase (CAT), mixed-function oxidase (MFO), carboxylesterases (CarE) and glutathione S-transferase (GST) activities in response to 24 h exposure of beetles to sublethal and lethal thymol concentrations. Our results showed that thymol decreased adult survival, longevity and percentage of adult emergence. Higher median lethal concentration (LC50) was recorded in females indicating their higher tolerance comparing to males. Overall, activities of SOD, CAT and CarE increased at sublethal and MFO increased at both sublethal and lethal thymol concentrations. On the other hand, GST and AChE activities decreased along with the increase in thymol concentrations from sublethal (1/5 of LC50, 1/2 of LC50) to lethal (LC50). Enzyme responses to the presence of thymol on bean seed were sex-specific. In the control group females had lower CarE and higher SOD, CAT and GST activity than males. In treatment groups, females had much higher CAT activity and much lower CarE activity than males. Our results contribute to deeper understanding of physiological mechanisms underlying thymol toxicity and tolerance which should be taken into account in future formulation of a thymol-based insecticide.

Biochemical Basis of Synergism between Entomopathogens and Insecticide Spinetoram against Plutella xylostella (L.)

Increasing incidences of insecticide resistance in diamondback moth Plutella xylostella (L.), the most ravaging pest of cruciferous vegetables, have stimulated interest in alternative pest management strategies. In this study, we have developed a spinetoram-resistant population of P. xylostella and examined the single and combined toxicity of Bacillus thuringiensis subsp. kurstaki and Beauveria bassiana with spinetoram. Additionally, enzyme activities were tested to study the influence of biopesticides in the mitigation of detoxifying enzymes. P. xylostella has developed high level of resistance (174.06-fold) against spinetoram after 16 generations of selection. B. thuringiensis subsp. kurstaki and B. bassiana exhibited high toxicity to spinetoram-resistant population of P. xylostella. Synergism was observed between B. thuringiensis subsp. kurstaki + spinetoram and B. bassiana + spinetoram with co-toxicity factor of 30.23 and 22.51, respectively. The joint application of spinetoram + B. bassiana and spinetoram + B. thuringiensis subsp. kurstaki resulted in suppression of mixed-function oxidase activity by 7.93 to 9.23 per cent and carboxyl-esterase activity by 5.64 to 6.18 per cent. Mixtures of biopesticides with spinetoram exhibited synergistic effects and may aid the design of new combinations of management strategies to delay resistant development in P. xylostella.

Bioefficacy of mosquito mat vaporizers and associated metabolic detoxication mechanisms in Aedes aegypti (Linnaeus) in Selangor, Malaysia: A statewide assessment.

This study aims to examine the efficacy of mosquito mat vaporizers on Aedes aegypti and their associated metabolic detoxication mechanisms. For this purpose, Aedes aegypti (Linnaeus) was collected from nine districts in Selangor, Malaysia and tested with mosquito vaporizing mat bioassays. The same populations were also subjected to biochemical assays to investigate activities of detoxifying enzymes, namely non-specific esterase (EST), glutathione-S-transferase (GST) and mixed function oxidase (MFO). The efficacy of Ae. aegypti on the active ingredients tested in decreasing order were d- allethrin > dimefluthrin > prallethrin with PBO > prallethrin. The results further indicated significant enhancement mean levels of EST, GST and MFO in pyrethroid-resistant populations. The mortality rate of Ae. aegypti in response to pyrethroid active ingredients was associated with MFO activity, suggesting it is an important detoxification enzyme for the populations tested. In view of the presence of resistance against household insecticide products, pyrethroid efficacy on Ae. aegypti populations needs to be monitored closely to ensure the implementation of an effective vector control program in Malaysia.

Cross-resistance, fitness costs, and biochemical mechanism of laboratory-selected resistance to tenvermectin A in Plutella xylostella.

Tenvermectin A is a new avermectin derivative that has good insecticidal and acaricidal effects. In order to study the resistance of Plutella xylostella to tenvermectin A, a sensitive strain (SS) and a laboratory-selected tenvermectin A-resistant strain (RS, 33.57-fold) were used to evaluate cross-resistance and fitness costs as well as to determine the resistance mechanism. There was no cross-resistance with common pesticides except for moderate cross-resistance with cypermethrin (resistance ratio = 10.26-fold) observed in RS. The activities of metabolic enzymes were measured, and the results showed that mixed function oxidase (MFO) and carboxylate esterase (CarE) in RS increased significantly by 2.92- and 2.86-fold, respectively, compared with SS. In addition, there was no obvious difference in glutathione-S-transferase (GST), which indicated that enhanced MFO and CarE activities may be the main mechanisms of detoxification. In the four typical resistance-related genes, expression of GluCl (4.86-fold), ABCC2 (3.85-fold), and CYP6 (2.94-fold) in RS were significantly promoted, but expression of GST was not. The clone and sequence of the PxGluClα subunit displayed six mutations that could lead to changes in the amino acid residues. High suitability related to tenvermectin A resistance was observed in RS, and it was found that the developmental stages of RS were significantly shortened and the survival rate of females was reduced. In addition, the mechanism of resistance to tenvermectin A may be regulated by the glutamate-gated chloride channel, ATP-binding cassette transporter, and MFO. In general, the study of resistance and biochemical mechanisms can provide beneficial and rational information for the management of resistance in P. xylostella. © 2021 Society of Chemical Industry.

Enhancement of Temephos and Deltamethrin Toxicity by Petroselinum crispum Oil and its Main Constituents Against Aedes aegypti (Diptera: Culicidae)

Previous work presented the profound antimosquito potential of Petroselinum crispum essential oil (PEO) against either the pyrethroid-susceptible or resistant strains of Aedes aegypti. This plant oil also inhibited the activity of acetylcholinesterase and mixed-function oxidases significantly, thus suggesting its potential as a synergist for improving mosquitocidal efficacy of insecticidal formulations. This study investigated the chemical composition, larvicidal activity, and potential synergism with synthetic insecticides of PEO and its main compounds for the purpose of interacting with insecticide resistance in mosquito vectors. The chemical profile of PEO, obtained by GC-MS analysis, showed a total of 17 bioactive compounds, accounting for 99.09% of the whole oil, with the most dominant constituents being thymol (74.57%), p-cymene (10.73%), and γ-terpinene (8.34%). All PEO constituents exhibited promising larvicidal effects, with LC50 values ranging from 19.47 to 59.75 ppm against Ae. aegypti, in both the pyrethroid-susceptible and resistant strains. Furthermore, combination-based bioassays revealed that PEO, thymol, p-cymene, and γ-terpinene enhanced the efficacy of temephos and deltamethrin significantly. The most effective synergist with temephos was PEO, which reduced LC50 values to 2.73, 4.94, and 3.28 ppb against MCM-S, PMD-R, and UPK-R, respectively, with synergism ratio (SR) values of 1.33, 1.38, and 2.12, respectively. The best synergist with deltamethrin also was PEO, which reduced LC50 values against MCM-S, PMD-R, and UPK-R to 0.008, 0.18, and 2.49 ppb, respectively, with SR values of 21.25, 9.00, and 4.06, respectively. This research promoted the potential for using essential oil and its principal constituents as not only alternative larvicides, but also attractive synergists for enhancing efficacy of existing conventional insecticides.

Toxicity and sublethal effects of chlorantraniliprole and indoxacarb on Spodoptera littoralis (Lepidoptera: Noctuidae)

Chlorantraniliprole and indoxacarb insecticides exhibit good efficiency for control lepidopteran pests. The current study is a comprehensive analysis of the effect of lethal and sublethal concentrations of these insecticides on Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae) by using the leaf dipping technique. The LC50 values ranged from 0.06 to 1.07 mg/L, and 0.005 to 0.81 mg/L for chlorantraniliprole and indoxacarb, respectively. Our results showed that the treatment of the 2nd instar larvae with LC50 concentrations of these insecticides significantly increased the length of larval and pupal duration as well as pupal weight in most cases. While, no significant differences have been found in the percentage of hatchability except for LC50 equivalent of indoxacarb. Female behavior regarding calling activity decreased by 50–60% following exposure to the LC50 concentration of both insecticides. Gas chromatography analysis results showed that both insecticides lowered pheromone titer except at chlorantraniliprole LC50 equivalent for (Z,E)-9,12-tetradecadien-l-ol acetate, and indoxacarb LC10 equivalent for (Z)-9-tetradecenyl acetate. Additionally, the activity of mixed-function oxidases and glutathione S-transferase were elevated relative to control. The carboxylesterase activity significantly increased when assayed with both chlorantraniliprole concentrations and indoxacarb LC10 equivalent. These results indicate that chlorantraniliprole and indoxacarb could be effective for S. littoralis control.

Culex erythrothorax (Diptera: Culicidae): Activity periods, insecticide susceptibility and control in California (USA).

The mosquito Culex erythrothorax Dyar is a West Nile virus (WNV) vector that breeds in wetlands with emergent vegetation. Urbanization and recreational activities near wetlands place humans, birds and mosquitoes in close proximity, increasing the risk of WNV transmission. Adult Cx. erythrothorax abundance peaked in a wetland bordering the San Francisco Bay of California (USA) during the first 3 hours after sunset (5527 ± 4070 mosquitoes / trap night) while peak adult Culex tarsalis Coquillett abundance occurred during the subsequent 3 h period (83 ± 30 Cx. tarsalis). When insecticide resistance was assessed using bottle bioassay, Cx. erythrothorax was highly sensitive to permethrin, naled, and etofenprox insecticides compared to a strain of Culex pipiens that is susceptible to insecticides (LC50 = 0.35, 0.71, and 4.1 μg/bottle, respectively). The Cx. erythrothorax were 2.8-fold more resistant to resmethrin, however, the LC50 value was low (0.68 μg/bottle). Piperonyl butoxide increased the toxicity of permethrin (0.5 μg/bottle) and reduced knock down time, but a higher permethrin concentration (2.0 μg/bottle) did not have similar effects. Bulk mixed-function oxidase, alpha-esterase, or beta-esterase activities in mosquito homogenates were higher in Cx. erythrothorax relative to the Cx. pipiens susceptible strain. There was no difference in the activity of glutathione S-transferase between the two mosquito species and insensitive acetylcholine esterase was not detected. Larvicides that were applied to the site had limited impact on reducing mosquito abundance. Subsequent removal of emergent vegetation in concert with larvicide applications and reduced daily environmental temperature substantially reduced mosquito abundance. To control Cx. erythrothorax in wetlands, land managers should consider vegetation removal so that larvicide can efficiently enter the water. Vector control agencies may more successfully control adult viremic Cx. erythrothorax that enter nearby neighborhoods by applying adulticides during the 3 h that follow sunset.

In Vivo Antibacterial Effect of Thymoquinone-Encapsulated Chitosan Nanoparticles in Bombyx mori L. Infected by Pathogenic Bacteria

The present work studied the antibacterial effect of different formulations of Chitosan (Chs); free Thymoquinone (Tq), and Thymoquinone-loaded chitosan nanoparticles (Tq-Chs NPs) on Bombyx mori L. infected by pathogenic bacteria, by evaluating some biochemical aspects of larval haemolymph. Bombyx mori larvae classified into three groups; each group was fed on mulberry leaves treated with one of the formulations (free Tq, free Chs and Tq-encapsulated Chs) with drug concentrations (0.05% and 0.1%). Before being used, the prepared formulations were characterized by size, morphology, encapsulation efficiency; in addition, in vitro drug release experiment was performed. The biochemical results revealed an improvement in the antibacterial and antioxidant activity of the studied formulations, especially with increasing the concentrations compared to control; (Tq-encapsulated Chs) followed by free Chs and free Tq revealed highly significant decreases in mixed function oxidase (MFO) activity, especially larvae treated with the high concentration (0.1%). The activity of acid phosphatase, total lipid content and carbohydrates pointed to a highly significant increase. Similarly, the qualitative analysis of silkworm proteins showed an obvious variation in the number and position of the detected protein bands from one treatment to another. It can be concluded that Tq and Chs showed an inhibitory impact against pathogenic bacteria infecting Bombyx mori larvae, proved their effects as antioxidant and anti-inflammatory agents which could be improved by loading Tq on Chs nanoparticles (Tq-Chs NPs).

Activities of Select Enzymes Involved in Insecticide Resistance in Spinosad-Resistant and -Susceptible Strains of Musca domestica (Diptera: Muscidae).

A Musca domestica L. strain collected from Pakistan has recently been shown to be resistant to spinosad; however, there is scarce information about the mechanism of resistance. For this reason, we explored whether a metabolic-based mechanism was responsible by analyzing the activities of the metabolic detoxifying enzymes, carboxylesterases, glutathione S-transferases, and mixed-function oxidases, in both a spinosad-selected (Spin-SEL) strain of M. domestica and a susceptible counterpart (Lab-susceptible). The results revealed that both strains were statistically at par in terms of enzyme activities. The activity of carboxylesterases in the Lab-susceptible strain was 78.17 ± 3.06 in comparison to 79.16 ± 3.31 nmol min-1 mg-1 in the Spin-SEL strain. The activity of mixed-function oxidases was 51.58 ± 4.20 in the Lab-susceptible strain, whereas 54.33 ± 4.08 pmol min-1 mg-1 was recorded in the Spin-SEL strain. The activity of glutathione S-transferases was 86.50 ± 4.59 (Lab-susceptible) and 90.33 ± 2.81 nmol min-1 mg-1 (Spin-SEL). These results revealed that the studied enzymes might not be responsible for spinosad resistance in the studied strain of M. domestica. Therefore, studies should be extended to find out other possible mechanisms of spinosad resistance.

Influence of Oilseed Rape Seed Treatment with Imidacloprid on Survival, Feeding Behavior, and Detoxifying Enzymes of Mustard Aphid, Lipaphis erysimi.

Imidacloprid application, as a seed coating agent on oilseed rape, is recommended to control mustard aphid, Lipaphis erysimi (Kaltenbach) (Hemiptera: Aphididae). In this study, responses of L. erysimi were investigated, including survival, feeding behavior, and detoxifying enzymes, on the oilseed rape seedlings grown from seeds coated with imidacloprid at rates of 6, 12, or 18 g active ingredient (a.i.)/kg seed. The results showed that the aphids’ survival rate, together with that of the progeny of the survivors, on the seed-treated seedlings significantly decreased. This indicates that the aphid population in fields can be suppressed effectively. The electrical penetration graph (EPG) technique was used to record aphid feeding behaviors on two-, four-, and six-leaf stages of oilseed rape seedlings that had been seed-coated with imidacloprid, and individual responses were revealed during the aphid feeding behavior. On the plants at the two-leaf stage, aphid feeding behaviors were influenced, showing decreased frequency of stylet penetration into the leaf (probe) or into the mesophyll cells (potential drops, pds for short), and shortened duration of stylet event in the leaf (probe) or in the phloem. On the plants at the four- and six-leaf stages, these impacts of imidacloprid were weakened; however, the saliva secretion duration in phloem was shortened to less than 5 min in all imidacloprid treatments. The activity of mixed-function oxidase in aphids maintained on the treated seedlings with imidacloprid was elevated. In conclusion, imidacloprid could be used as a seed coating agent for aphid control, but chemical resistance in aphids should not be ignored.

Effect of cypermethrin on worker and soldier termites of subterranean termites Odontotermes brunneus (Hagen) (Termitidae: Isoptera)

The termite Odontotermes brunneus is an economically important species causing damage to cellulose containing wooden material and agricultural crops in India. Insecticide application is an effective strategy in termite control. In the present study the effect of cypermethrin was tested for workers and soldiers termite using filter paper dip method. After 24 h treatment the lethal concentration (LC50) was increased to 9.7 ppm in workers and 1.8 ppm in soldiers respectively. The detoxification enzyme activities of esterase, glutathione S-transferase was increased in worker termites 23 µmol, 9 µmol/min/mg of protein compared to soldiers 15 µmol, 7 µmol/min/mg of protein respectively (p < 0.05). The activity of mixed-function oxidase was found very less in both samples. Further nPAGE analysis revealed that increased esterase band in workers than soldier and control sample. The data of this study revealed that possible mechanism of esterase and glutathione S-transferase mediated cypermethrin detoxification that leads to reduce the sensitivity in worker termites of O. brunneus.

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.

Modulation in Activity Profiles in Insecticide-Resistant Population of Tobacco Caterpillar, &lt;i&gt;Spodoptera Litura&lt;/i&gt; (Fabricius)

Activity spectrum of detoxification enzymes was systematically assessed in tobacco caterpillar, Spodoptera litura collected from four locations in Kerala, India, to decipher the mechanism of insecticide resistance. Using the susceptible check ICAR-NBAIR strain, specific activity profiles of acetylcholine esterase (AChE) were found to be 16.16-, 10.71- and 4.88-fold higher in the Kovilnada, Palappur and Kanjikuzhi populations respectively. Specific activities of mixed function oxidase (MFO) were also found to be 19.24-, 17.11-, 6.08-fold higher in the same populations respectively, indicating the predominance of AChE and MFO towards imparting resistance. Carboxylesterase (CarE) and glutathion-S-transferase (GST) specific activity profiles were 3.62- and 3.37-fold higher in the Kovilnada population, followed by 2.89- and 2.98-fold higher in the Palappur population and as 2.10- and 1.15-fold higher in the Kanjikuzhi population, indicating their partial role in resistance development. Suppression of specific activities in synergism bioassays with AChE in chlorpyriphos + TPP treatment (9.32-fold), GST in chlorpyriphos + DEM (4.78-fold) and CarE in quinalphos + TPP (5.15-fold) highlighted the involvement of multiple detoxification enzymes conferring resistance to organophosphates. Reduced activity of MFO in case of lambda-cyhalothrin + PBO (5.35-fold), CarE in case of cypermethrin + TPP (7.36-fold) and 3.60-fold reduction in MFO in case of cypermethrin + PBO highlighted the role of esterases and MFOs towards resistance development against synthetic pyrethroids.

Pyrethroids Use: Threats on Metabolic-Mediated Resistance Mechanisms in the Primary Dengue Vector Aedes aegypti (Diptera: Culicidae)

The emergence of pyrethroid resistance in Aedes aegypti (L.) has limited the success of vector control. Early detection of resistance could assist authorities in deciding well-suited control strategies to minimize operational failures of Ae. aegypti control. Herein, biochemical analysis was performed to investigate the mechanisms involved in pyrethroid resistance in nine populations of Indonesian Ae. aegypti. Enzymes of adult Ae. aegypti such as esterases (ESTs), glutathione-S-transferases (GSTs), and mixed-function oxidases (MFOs) were characterized. Elevated MFO activity was correlated with resistance phenotype, indicating the role of this enzyme in contributing to pyrethroid resistance. No significant correlations were shown between pyrethroid resistance phenotype and α-ESTs, suggesting that marginally exceeded enzyme levels relative to the reference strain in some pyrethroid-susceptible populations were causative factor for insecticide resistance in other groups of insecticides. However, significant correlation was demonstrated between β-ESTs and pyrethroid resistance phenotype. The lowest enzyme levels in GSTs indicated that this enzyme was not predominant in causing pyrethroid resistance, despite the presence of significant correlations. Because metabolic detoxification fails to comprehensively explain the pyrethroid resistance in some Indonesian Ae. aegypti, additional mechanisms such as altered target sites in voltage-gated sodium channel may also contribute to the high pyrethroid resistance in Ae. aegypti.

Protective and Detoxifying Enzyme Activity and ABCG Subfamily Gene Expression in Sogatella furcifera Under Insecticide Stress

Sogatella furcifera, an important migratory pest of rice, has substantial detrimental effects on rice production. To clarify the mechanism whereby S. furcifera responds to insecticide stress, we measured the activity of its protective [superoxide dismutase (SOD); peroxidase (POD); catalase (CAT)] and detoxifying [carboxylesterase (CarE); glutathione S-transferase (GST); mixed-function oxidase (MFO)] enzymes and the expression levels of its ATP-binding cassette subfamily G (ABCG) transporter genes in response to sublethal concentrations (LC10 and LC25) of the insecticides thiamethoxam, buprofezin, and abamectin. On the bases of the transcriptome data and the ABCG genes of Laodelphax striatellus, we obtained 14 full-length ABCG sequences for S. furcifera. RT-qPCR results showed that 13, 12, and 9 sfABCG genes were upregulated in the presence of thiamethoxam, buprofezin, and abamectin, respectively, at LC10. Moreover, 13 and 7 sfABCG genes were upregulated following treatment with thiamethoxam and abamectin, respectively, at LC25. Enzyme activity assays showed that although thiamethoxam, buprofezin, and abamectin induced GST, CarE, CAT, POD, and SOD activity, they did so at different concentrations and exposure times. The activity of MFO was generally inhibited with prolonged exposure to the three insecticides, with the inhibitory effect being most significant at 72 h. These results indicate that S. furcifera differs in its response to different types or concentrations of insecticides. Taken together, our results lay the foundations for gaining a deeper understanding of the mechanisms underlying the adaptation of S. furcifera to different types of insecticides, which would be of considerable significance for the development of effective pest management strategies.

Biochemical Effects of Petroselinum crispum (Umbellifereae) Essential Oil on the Pyrethroid Resistant Strains of Aedes aegypti (Diptera: Culicidae).

In ongoing screening research for edible plants, Petroselinum crispum essential oil was considered as a potential bioinsecticide with proven antimosquito activity against both the pyrethroid susceptible and resistant strains of Aedes aegypti. Due to the comparative mosquitocidal efficacy on these mosquitoes, this plant essential oil is promoted as an attractive candidate for further study in monitoring resistance of mosquito vectors. Therefore, the aim of this study was to evaluate the impact of P. crispum essential oil on the biochemical characteristics of the target mosquito larvae of Ae. aegypti, by determining quantitative changes of key enzymes responsible for xenobiotic detoxification, including glutathione-S-transferases (GSTs), α- and β-esterases (α-/β-ESTs), acetylcholinesterase (AChE), acid and alkaline phosphatases (ACP and ALP) and mixed-function oxidases (MFO). Three populations of Ae. aegypti, comprising the pyrethroid susceptible Muang Chiang Mai-susceptible (MCM-S) strain and the pyrethroid resistant Pang Mai Dang-resistant (PMD-R) and Upakut-resistant (UPK-R) strains, were used as test organisms. Biochemical study of Ae. aegypti larvae prior to treatment with P. crispum essential oil revealed that apart from AChE, the baseline activity of most defensive enzymes, such as GSTs, α-/β-ESTs, ACP, ALP and MFO, in resistant UPK-R or PMD-R, was higher than that determined in susceptible MCM-S. However, after 24-h exposure to P. crispum essential oil, the pyrethroid susceptible and resistant Ae. aegypti showed similarity in biochemical features, with alterations of enzyme activity in the treated larvae, as compared to the controls. An increase in the activity levels of GSTs, α-/β-ESTs, ACP and ALP was recorded in all strains of P. crispum oil-treated Ae. aegypti larvae, whereas MFO and AChE activity in these mosquitoes was decreased. The recognizable larvicidal capability on pyrethroid resistant Ae. aegypti, and the inhibitory effect on AChE and MFO, emphasized the potential of P. crispum essential oil as an attractive alternative application for management of mosquito resistance in current and future control programs.