P450 Monooxygenase Activity Research Articles

Rational design and synthesis of new pyrrolone candidates as prospective insecticidal agents against Culex pipiens L. Larvae

As a result of its high reactivity, furan-2(3H)-one derivative 2 can be selected as a versatile and suitable candidate for building of novel nitrogen heterocyclic compounds. Consequently, furan-2(3H)-one derivative 2 and some nitrogen nucleophiles were utilized as starting materials for the formation of new pyridazinone and pyrrolone derivatives bearing naphthalene moiety. The continuous buildup of insecticide resistance is the main obstacle facing pest control measures. Pyrrole-based insecticides are a favourable choice due to their unique mode of action and no cross-resistance with traditional neurotoxic insecticides. The larvicidal activities of pyrrolone derivatives were assessed against field and laboratory strains of Culex pipiens larvae in comparison with chlorfenapyr (pyrrole insecticide). Compounds 17 (21.05 µg/mL) > 9 (22.81 µg/mL) > 15 (24.39 µg/mL) > 10 (26.76 µg/mL) > 16 (32.09 µg/mL) were most effective against lab strain of C. pipiens larvae relative to chlorfenapyr (25.43 µg/mL). While in field strain, 17 and 15 were the most toxic compounds followed by 9 > 10 > 16 > 2 with LC50 of 9.87, 10.76, 11.52, 12.68, 15.32 and 18.37 µg/mL, respectively, compared with chlorfenapyr with 14.03 µg/mL. The cytochrome P-450 monooxygenase activities were significantly increased in treated larvae of lab and field strains relative to untreated. The great variations in toxicity of the synthesized compounds were interpreted by structure-activity relationship study. The pyrrolone derivatives are effective against field and insecticide-resistant strains. Therefore, they are considered promising compounds to be integrated into pest management programs.

Stability, Inheritance, Cross-Resistance, and Fitness Cost of Resistance to λ-Cyhalothrin in Cydia pomonella.

Insecticides are commonly utilized in agriculture and forestry for pest control, but their dispersal can pose hazards to humans and environment. Understanding resistance, inheritance patterns, and fitness costs can help manage resistance. A λ-cyhalothrin-resistant population (LCR) of Cydia pomonella, a global pest of pome fruits and walnuts, was obtained through selective insecticide breeding for 15 generations, showing stable moderate resistance (23.85-fold). This population was cross-resistant to deltamethrin (4.26-fold) but not to β-cypermethrin, chlorantraniliprole, chlorpyrifos, and avermectin. Genetic analysis revealed the resistance was autosomal, incompletely dominant, and controlled by multiple genes. Increased activity of glutathione S-transferases and cytochrome P450 monooxygenases (P450s) played a primary role in resistance, with specific genes up-regulated in LCR, and exhibited significant expression in midgut. LCR also exhibited fitness costs, including delays in development, reduced fecundity, and slower population growth. These findings contribute to understanding λ-cyhalothrin resistance in C. pomonella and can guide resistance management strategies.

Resistance monitoring of diamide insecticides and characterization of field-evolved chlorantraniliprole resistance among Chinese populations of the tomato pinworm Phthorimaea (=Tuta) absoluta (Lepidoptera: Gelechiidae)

The tomato pinworm, Phthorimaea (=Tuta) absoluta, is considered one of the most destructive and invasive insect pests worldwide, having developed significant resistance to many popular insecticides. In this study, we monitored the field resistance of P. absoluta populations from China to three diamide insecticides: flubendiamide, chlorantraniliprole, and cyantraniliprole. We found that one field population from Wuzhong City (WZ) exhibited high level of resistance to chlorantraniliprole. Using the WZ population and a susceptible reference strain (YN-S), we established a near-isogenic line (WZ-NIL) of P. absoluta with resistance to chlorantraniliprole. This strain also showed substantial cross-resistance to flubendiamide, and cyantraniliprole. Genetic analysis revealed that the inheritance of resistance to chlorantraniliprole in the WZ-NIL strain was autosomal and incompletely dominant. Additionally, the pesticide synergist piperonyl butoxide significantly inhibited chlorantraniliprole resistance by compromising P450 monooxygenase activity, which was significantly higher in the resistant strain. Furthermore, WZ-NIL had significantly prolonged developmental stages, lower pupation rates, reduced female fecundity, and lower egg hatchability than YN-S individuals. The fitness of WZ-NIL relative to YN-S was estimated to be 0.73, indicating significant fitness cost associated with chlorantraniliprole resistance. Rotating chlorantraniliprole with other insecticides that have different modes of action and degradation may be particularly useful for managing chlorantraniliprole resistance in P. absoluta.

Dietary oxyclozanide influences antioxidant enzyme activities and damages DNA in Galleria mellonella (Lepidoptera: Pyralidae).

Oxyclozanide (OXY) is an anthelmintic widely used in the treatment of flatworm infection and fasciolosis. It also has antiadenovirus, antibiofilm, antifungal, and antibacterial activities. Various chemicals have been suggested as alternative chemicals in insect pest management. Here, the oxidative and genotoxic effects of OXY on 7th instars, pupae and adults of the model organism Galleria mellonella (Linnaeus) (Lepidoptera: Pyralidae) were examined. First-instar larvae were reared on 0.003, 0.03, 0.3, and 1.5 g OXY per 100 g artificial diets. Compared with all tested OXY concentrations and controls without OXY, dietary OXY led to increased antioxidant capacity and genotoxic effects. Concentrations of malondialdehyde, an oxidative stress marker, were significantly increased in adults of larvae reared on OXY-charged diets at 0.3 and 1.5 g/100 g compared to the adult control group. We also recorded a significant increase in the genotoxic test data (Tail length, Tail DNA %, Tail moment) at the same stages and concentrations. We recorded significant increases in glutathione-S-transferase, superoxide dismutase (SOD) and glutathione peroxidase activities in larvae fed high OXY concentrations. SOD and catalase activities were also significantly increased at the concentration of 0.03 g/100 g of OXY in the pupal and adult stages. Cytochrome P450 monooxygenase activity was significantly increased at the highest concentration of OXY in the larval and pupal stages. Also, our regression analysis indicates a correlation between the markers of oxidative stress, antioxidant enzymes and comet parameters. These data indicate that OXY induces oxidative stress and antioxidative enzyme response.

Comparative study on biochemical responses to imidacloprid and clothianidin in cockroaches (Blattella germanica)

AbstractCockroaches, ubiquitous pests known for significant reproduction and as potential vectors of diseases, are notorious for their adoptability to a variety of insecticides that cause substantial challenges in pest control. The present research evaluated the LC50 concentrations of imidacloprid (0.65 mg/L) and clothianidin (2.5 μg/L) for Blattella germanica using topical and bait methods. The cockroaches were exposed to one‐third concentration of LC50 at a regular interval of 1, 2, 4, 8, 12, 16 and 20 days. During exposure, the activity of P450 monooxygenase increased, whereas the activity of brain acetylcholinesterase decreased. Lactate dehydrogenase, alkaline and acid phosphatase activity were highest on day 4, with a slight recovery on day 20. The elevated activities for superoxide dismutase and catalase, as well as increased lipid peroxidation levels on day 4, followed by a significant recovery in activity on day 20. The glutathione‐S‐transferase activity was elevated while the glutathione content was reduced, and a significant restoration was observed at the end of the experiment. The biochemical mechanisms of the organisms such as P450, esterase enzymes and oxidative systems, are actively involved in the detoxification mechanism. The study elucidated that the insecticide defence mechanism triggers detoxification pathways that encompass the metabolism of endogenous insecticidal compounds, with clothianidin showing greater efficacy and lower detoxification compared with imidacloprid.

The P450-Monooxygenase Activity and CYP6D1 Expression in the Chlorfenapyr-Resistant Strain of Musca domestica L.

The house fly Musca domestica L. is one of the most common insects of veterinary and medical importance worldwide; its ability to develop resistance to a large number of insecticides is well known. Many studies support the involvement of cytochrome P-450-dependent monooxygenases (P450) in the development of resistance to pyrethroids, neonicotinoids, carbamates, and organophosphates among insects. In this paper, the monooxygenase activity and expression level of CYP6D1 were studied for the first time in a chlorfenapyr-resistant strain of house fly. Our studies demonstrated that P450 activity in adults of the susceptible strain (Lab TY) and chlorfenapyr-resistant strain (ChlA) was 1.56-4.05-fold higher than that in larvae. In females of the Lab TY and ChlA strains, this activity was 1.53- and 1.57-fold higher, respectively (p < 0.05), than that in males, and in contrast, the expression level of CYP6D1 was 21- and 8-fold lower, respectively. The monooxygenase activity did not vary between larvae of the susceptible strain Lab TY and the chlorfenapyr-resistant strain ChlA. Activity in females and males of the ChlA strain exceeded that in the Lab TY strain specimens by 1.54 (p = 0.08) and 1.83 (p < 0.05) times, respectively, with the same level of CYP6D1 expression. PCR-RFLP analysis revealed a previously undescribed mutation in the promoter region of the CYP6D1 gene in adults of the Lab TY and ChlA strains, and it did not affect the gene expression level. The obtained results show that the development of resistance to chlorfenapyr in M. domestica is accompanied by an increase in P450-monooxygenase activity without changes in CYP6D1 expression.

Baseline of susceptibility, risk assessment, biochemical mechanism, and fitness cost of resistance to dimpropyridaz, a novel pyridazine pyrazolecarboxamide insecticide, in Bemisia tabaci from China

Bemisia tabaci is one of the most destructive agricultural insect pests around the world, and it has developed high levels of resistance to most pesticides. Dimpropyridaz, a novel insecticide developed by BASF, displays excellent activity against piercing-sucking insect pests. In this study, baseline of susceptibility showed all tested field populations of B. tabaci are susceptible to dimpropyridaz. After continuous selection with dimpropyridaz in the lab, a B. tabaci strain (F12) developed 2.2-fold higher level of resistance compared with a susceptible MED-S strain, and the realized heritability (h2) was estimated as 0.0518. The F12 strain displayed little cross-resistance to afidopyropen, cyantraniliprole, sulfoxaflor, or abamectin, and significantly increased activity of cytochrome P450 monooxygenase (P450). The fitness cost of dimpropyridaz resistance was evident in F12 strain, which had a relative fitness of 0.95 and significantly lower fecundity per female compared with MED-S strain. Taken together, B. tabaci displays high susceptibility to dimpropyridaz in the field, and low risk of developing resistance to dimpropyridaz under successive selection pressure. Little cross-resistance to popular insecticides was found, and fitness cost associated dimpropyridaz resistance was observed. Higher activity of cytochrome P450 in the F12 strain, may be involved in the process of detoxifying dimpropyridaz in whitefly.

Synergistic action and mechanism of scoparone, a key bioactive component of Artemisia capillaris, and spirodiclofen against spider mites.

Plants have numerous defensive secondary metabolites to withstand insect attacks. Scoparone, which is extracted from the medicinal plant Artemisia capillaris, has potent acaricidal effects on Tetranychus cinnabarinus. Spirodiclofen, derived from a tetronic acid derivative, is a potent commercial acaricide that is extensively used globally. However, whether scoparone has synergistic effects when used in conjunction with spirodiclofen and the underlying synergistic mechanism remains unclear. Scoparone exhibited a potent synergistic effect when it was combined with spirodiclofen at a 1:9 ratio. Subsequently, cytochrome P450 monooxygenase (P450) activity, RNA-Seq and qPCR assays indicated that the enzyme activity of P450 and the expression of one P450 gene from T. cinnabarinus, TcCYP388A1, were significantly inhibited by scoparone and spirodiclofen + scoparone; conversely, P450 was activated in spirodiclofen-exposed mites. Importantly, RNAi-mediated silencing of the TcCYP388A1 gene markedly increased the susceptibility of spider mites to spirodiclofen, scoparone and spirodiclofen + scoparone, and in vitro, the recombinant TcCYP388A1 protein could metabolize spirodiclofen. Molecular docking and functional analyses further indicated that R117, which is highly conserved in Arachnoidea species, may be a vital specific binding site for scoparone in the mite TcCYP388A1 protein. This binding site was subsequently confirmed using mutagenesis data, which revealed that this binding site was the sole site selected by scoparone in spider mites over mammalian or fly CYP388A1. These results indicate that the synergistic effects of scoparone and spirodiclofen on mites occurs through the inhibition of P450 activity, thus reducing spirodiclofen metabolism. The synergistic effect of this potent natural product on the detoxification enzyme-targeted activity of commercial acaricides may offer a sustainable strategy for pest mite resistance management. © 2024 Society of Chemical Industry.

Risk assessment, cross-resistance, biochemical mechanism and fitness cost of tetraniliprole resistance in the tomato pinworm Tutaabsoluta

Tuta absoluta is one of the most devastating and invasive insect pests throughout the world. It feeds on numerous solanaceous plant species and has evolved resistance to various types of popular chemical agents. Tetraniliprole is a novel diamide chemical agent that acts as a modulator of the ryanodine receptor, and it has been considered as a promising insecticide against T. absoluta. In this study, we subjected a T. absoluta strain to 17 generations of continuous selection with tetraniliprole in the lab. The resulting tetraniliprole-selected T. absoluta strain (YN-Tet) exhibited a 12.9-fold higher resistance to tetraniliprole compared with a susceptible strain (YN–S), and the realized heritability (h2) was estimated to be 0.1437. The YN-Tet strain exhibited a low level of cross-resistance to chlorantraniliprole and significantly higher cytochrome P450 monooxygenase activity. There was a fitness cost of tetraniliprole resistance in the YN-Tet strain, which had a relative fitness of 0.83 compared with the YN-S strain and a significantly lower fecundity per female. These results enhance our knowledge of the risk and mechanism of tetraniliprole resistance, enabling future optimization of resistance management strategies.

Knockdown of one cytochrome P450 gene CYP6DW4 increases the susceptibility of Bemisia tabaci to dimpropyridaz, a novel pyridazine pyrazolecarboxamide insecticide

Bemisia tabaci is a formidable insect pest worldwide, and it exhibits significant resistance to various insecticides. Dimpropyridaz is a novel pyridazine pyrazolecarboxamide insecticide used against sucking insect pests, but there is little information regarding its metabolic detoxification in arthropods or cross-resistance with other insecticides. In this study, we found that dimpropyridaz shows no cross-resistance with three other popular insecticides, namely abamectin, cyantraniliprole, and flupyradifurone. After treatment of B. tabaci adults with a high dose of dimpropyridaz, higher cytochrome P450 monooxygenase (P450) activity was detected in the survivors, and the expression of the P450 gene CYP6DW4 was highly induced. Cloning and characterization of the full-length amino acid sequence of CYP6DW4 indicated that it contains conserved domains typical of P450 genes, phylogenetic analysis revealed that it was closely related to a B. tabaci protein, CYP6DW3, known to be involved in detoxification of imidacloprid. Silencing of CYP6DW4 by feeding insects with dsRNA significantly increased the susceptibility of B. tabaci to dimpropyridaz. In addition, homology modeling and molecular docking analyses showed the stable binding of dimpropyridaz to CYP6DW4, with binding free energy of −6.65 kcal/mol. Our findings indicate that CYP6DW4 plays an important role in detoxification of dimpropyridaz and possibly promotes development of resistance in B. tabaci.

Risk of resistance and the metabolic resistance mechanism of Laodelphax striatellus (Fallén) to cyantraniliprole

Cyantraniliprole is a highly effective diamide insecticide used to control of Laodelphax striatellus (Fallén). This study aimed to assess the insecticide resistance risk of L. striatellus and its metabolic resistance mechanisms. After 25 continuous generations of selection, the resistance of L. striatellus to cyantraniliprole increased by 17.14-fold. The realistic heritability of resistance was 0.0751. After successive rearing for five generations without exposure to insecticides, the resistance ratio for the resistant strain of L. striatellus decreased by 3.47-fold, and the average resistance decline rate per generation was 0.0266. Cyantraniliprole-resistant strains did not exhibit cross-resistance to triflumezopyrim, pymetrozine, flonicamid, sulfoxaflor, dinotefuran, clothianidin, thiamethoxam, nitenpyram, or imidacloprid. Compared to those of the sensitive strain, the 2nd, 3rd, and 4th instars, nymphal stage durations, total preoviposition period, and average generation time of the resistant strain were markedly reduced. Furthermore, the activity of cytochrome P450 monooxygenase (P450) and carboxylesterase (CarE) were markedly increased. The upregulation of CYP419A1v2 expression was most evident among the P450 genes, with a 6.10-fold increase relative to that in the sensitive strain. The CarE gene LsCarE5 was significantly upregulated by 1.94-fold compared with that in the sensitive strain. With the continuous use of cyantraniliprole, L. striatellus may develop resistance to this insecticide. This resistance may be related to the increase in metabolic enzyme activities regulated by the overexpression of P450 and CarE genes.

Oxidative and genotoxic effect of piperazine on Galleria mellonella (Lepidoptera: Pyralidae) hemolymph.

Recently, there are many studies suggesting antibacterial, antifungal, and anthelmintic agents as alternative chemicals to insecticides. In this study, the oxidative and genotoxic effect of Piperazine, a clinically important hexahydropyrazine anthelmintic, on Galleria mellonella L. hemolymph tissue by adding artificial diet were investigated. Galleria mellonella larvae were reared until 7th larval stage in artificial diet containing 0.001, 0.01, 0.1, and 1 g piperazine per 100 g of diet. Using hemolymph collected from 7th-instar larvae, the amount of lipid peroxidation final product malondialdehyde (MDA), protein oxidation product protein carbonyl (PCO), and detoxification enzymes glutathione S-transferase (GST) and cytochrome P450 monooxygenase (cyt P450) activity, comet assay were measured. According to the results obtained, when the piperazine high concentrations tested with the control group were compared, statistically significant differences were found in MDA, PCO content, cyt P450, GST activity, and comet assay in the hemolymph of the insect. While MDA content was 0.01 ± 0.0021 nmol/mg protein in the control group, this amount increased approximately 2-fold at the highest concentration (0.0231 ± 0.0050 nmol/mg protein). On the other hand, when the control group and the highest piperazine concentration were compared in the GST and cyt P450 activity, it was determined that there was a statistically significant increase. We obtained similar results in comet assay and micronucleus formation data. This study showed that the tested piperazine concentrations caused significant changes in the detoxification capacity, oxidative stress, and genotoxic markers in the insect's hemolymph tissue.

Baseline Susceptibility, Cross-Resistance, and Sublethal Effects of Broflanilide, a Novel Meta-Diamide Pesticide, in Spodoptera litura

Spodoptera litura is a damaging and notorious insect pest of agricultural crops that has developed resistance to various insecticides. Broflanilide is a novel pesticide with a unique mode of action that displays high efficiency against lepidopterous larvae. We here determined the baseline susceptibility of a laboratory strain of S. litura to broflanilide and 10 other popular insecticides. Furthermore, we measured susceptibility and cross-resistance using three common insecticides in 11 field-collected S. litura populations. Broflanilide caused the highest toxicity among all tested insecticides, with the laboratory strain and all field-collected populations showing high susceptibility. Moreover, no cross-resistance was detected between broflanilide and the other tested insecticides. We subsequently evaluated the sublethal effects of broflanilide and found that treatment with the 25% lethal concentration (LC25) prolonged the development duration in the larvae, reduced the pupation rate and pupae weight, and decreased egg hatchability. Finally, the activities of three detoxifying enzymes were measured in S. litura after treatment with the LC25 dose. The results suggested that enhanced cytochrome P450 monooxygenase (P450) activity could be involved in broflanilide detoxification. Overall, these findings demonstrate the strong toxicity and significant sublethal effects of broflanilide in S. litura and suggest that increased P450 activity may be associated with broflanilide detoxification.

Enhancing the Toxicity of Cypermethrin and Spinosad against Spodoptera littoralis (Lepidoptera: Noctuidae) by Inhibition of Detoxification Enzymes.

The extensive use of wide-ranging insecticides in agricultural activities may develop resistance in insects. The dipping technique was utilized for examining changes in detoxifying enzyme levels in Spodoptera littoralis L. induced by cypermethrin (CYP) and spinosad (SPD) with and without a combination of three enzyme inhibitors: triphenyl phosphate (TPP), diethyl maleate (DEM), and piperonyl butoxide (PBO), at 70 μg/mL. PBO, DEM, and TPP showed 50% mortality against larvae at 236.2, 324.5, and 245.8 μg/mL, respectively. The LC50 value of CYP on S. littoralis larvae reduced from 2.86 μg/mL to 1.58, 2.26, and 1.96 μg/mL, while the LC50 value of SPD declined from 3.27 μg/mL to 2.34, 2.56, and 2.53, with the addition of PBO, DEM, and TPP, respectively, 24 h after treatment. Moreover, the activity of carboxylesterase (CarE), glutathione S-transferase (GST), and cytochrome P450 monooxygenase (Cyp 450) was significantly inhibited (p < 0.05) by TPP, DEM, PBO plus CYP, and SPD in S. littoralis larvae in comparison with tested insecticides alone. These findings suggested that three enzyme inhibitors play a major role in increasing the toxicity of CYP and SPD in S. littoralis and will provide insight into how to overcome insecticide resistance in insects.

Metabolic resistance to deltamethrin is mediated by P450 and esterases in common bed bugs Cimex lectularius L. (Heteroptera: Cimicidae)

The infestations of Cimex lectularius L. (Heteroptera: Cimicidae) registered in the last decades have been influenced by several human activities, including international tourism and commerce. Moreover, the development of insecticide resistance and careless pest control strategies contributed to the dispersal of bed bugs. Given the complexity of the topic, distinguishing physiological and molecular mechanisms involved in resistance can help design proper control tools and limit the resistance spread. Here we determined the susceptibility to deltamethrin and imidacloprid in bed bugs collected in Italy. Also, we assessed the role of esterases and P450 monooxygenases by direct enzymatic activity measurement and inhibition by synergism bioassays. Our results showed that the field-collected colony exhibited high resistance ratios to imidacloprid and deltamethrin (757 and &gt;60,000 times, respectively) compared to the susceptible colony. Moreover, resistant bed bugs showed increased activity of esterases and P450 monooxygenases. The synergistic effect of piperonyl butoxide (PBO) suggests the significant contribution of both enzymatic groups as detoxification pathways implicated in pyrethroid-resistant bed bugs. Further investigations are needed to unravel the biochemical and molecular basis involved in the resistant phenotype for developing novel strategies for pest control.

Resistance status and activity of detoxifying enzymes in Oligonychus coffeae (Nietner)(Acari:Tetranychidae) on tea

Regular monitoring of acaricide resistance in economically important plant feeding mites is important for effective management. Oligonychus coffeae is one of the most important mite pests of tea and acaricides are frequently used to manage this pest, yet control failures sometimes occur at recommended doses. Hence, bioassay experiments were conducted to confirm whether such control failures were caused by resistance development in populations collected from major tea growing areas of Sub-Himalayan West Bengal, India. The adult females of O. coffeae were bio-assayed with commonly used six acaricides viz., dicofol, ethion, fenpropathrin, fenpyroximate, fenazaquin and propargite. Moreover, we estimated the activity of general esterase (GE), glutathione S-transferase (GST) and cytochrome P450 monooxygenase in the field collected samples as well as laboratory reared susceptible population. Based on the LC50 values, the resistance ratio of the field collected populations compared to the laboratory reared susceptible population varied from 3.29 to 5.85-fold,4.13–11.21-fold,12.07–28.28-fold,1.19–1.67-fold,1.12–1.77-fold, 2.73–5.34-fold for dicofol, ethion, fenpropathrin, fenpyroximate, fenazaquin and propargite, respectively. General esterase, GST and MFO activities of the tested populations were in the ranges of 5.69–16.54 mOD/min/mg protein, 91.51–319.91 μM/min/mg protein and 0.040–0.071 mOD/min/mg protein, respectively. The highest enzyme activity was detected in the Naxalbari population which was one of the most resistant populations to all the tested acaricides. The findings will be useful to the tea industry for selection of most effective chemical for successful management of this pest. Use of fenpropathrin and ethion should be discontinued for tea pest management and use of other chemicals having different mode of actions may be continued with rotation.

Resistance selection of triflumezopyrim in Laodelphax striatellus (fallén): Resistance risk, cross-resistance and metabolic mechanism.

The risk assessment and resistance mechanisms of insecticide resistance are critical for resistance management strategy before a new insecticide is widely used. Triflumezopyrim (TFM) is the first commercialized mesoionic insecticide, which can inhibit nicotinic acetylcholine receptor with high-performance against the small brown planthopper (SBPH), Laodelphax striatellus (Fallén). In our study, the resistance of SBPH to TFM increased 26.29-fold, and the actual heritability of resistance was 0.09 after 21 generations of continuous selection by TFM. After five generations of constant feeding under insecticide-free conditions from F16 generation, the resistance level decreased 2.05-fold, and the average resistance decline rate per generation was 0.01, but there were no statistical decline. The TFM resistant strains had no cross-resistance to imidacloprid, nitenpyram, thiamethoxam, dinotefuran, flonicamid, pymetrozine, and chlorfenapyr. The third and fifth nymphal stage duration, pre-adult stage, adult preoviposition period, longevity, emergence rate, and hatchability of the resistant strain were significantly lower than those of the susceptible strain, while the female-male ratio was considerably increased. The fitness cost was 0.89. Further, cytochrome P450 monooxygenase (P450) and carboxylesterase (CarE) activities were markedly increased, but only the enzyme inhibitor piperonyl butoxide (PBO) had a significant synergistic effect on the resistant strain. The expression of CYP303A1, CYP4CE2, and CYP419A1v2 of P450 genes was significantly increased. SBPH has a certain risk of resistance to TFM with continuous application. The TFM resistance may be due to the increased activity of P450 enzyme regulated by the overexpression of P450 genes.

The Cross-Resistance Pattern and the Metabolic Resistance Mechanism of Acetamiprid in the Brown Planthopper, Nilaparvata lugens (Stål).

Acetamiprid is widely used in paddy fields for controlling Nilaparvata lugens (Stål). However, the risk of resistance development, the cross-resistance pattern and the resistance mechanism of acetamiprid in this pest remain unclear. In this study, an acetamiprid-resistant strain (AC-R) was originated from a field strain (UNSEL) through successive selection with acetamiprid for 30 generations, which reached 60.0-fold resistance when compared with a laboratory susceptible strain (AC-S). The AC-R strain (G30) exhibited cross-resistance to thiamethoxam (25.6-fold), nitenpyram (21.4-fold), imidacloprid (14.6-fold), cycloxaprid (11.8-fold), dinotefuran (8.7-fold), sulfoxaflor (7.6-fold) and isoprocarb (8.22-fold), while there was no cross-resistance to etofenprox, buprofezin and chlorpyrifos. Acetamiprid was synergized by the inhibitor piperonyl butoxide (2.2-fold) and the activity of cytochrome P450 monooxygenase was significantly higher in the AC-R strain compared with the AC-S strain, suggesting the critical role of P450. The gene expression results showed that the P450 gene CYP6ER1 was significantly overexpressed in AC-R compared with the AC-S and UNSEL strains. In addition, the RNA interference (RNAi) of CYP6ER1 significantly increased the susceptibility of AC-R to acetamiprid. Molecular docking predicted that acetamiprid and CYP6ER1 had close binding sites, and the nitrogen atoms had hydrogen bond interactions with CYP6ER1. These results demonstrated that the overexpression of CYP6ER1 contributed to acetamiprid resistance in N. lugens.

Characterization of a Virally Encoded Flavodoxin That Can Drive Bacterial Cytochrome P450 Monooxygenase Activity.

Flavodoxins are small electron transport proteins that are involved in a myriad of photosynthetic and non-photosynthetic metabolic pathways in Bacteria (including cyanobacteria), Archaea and some algae. The sequenced genome of 0305φ8-36, a large bacteriophage that infects the soil bacterium Bacillus thuringiensis, was predicted to encode a putative flavodoxin redox protein. Here we confirm that 0305φ8-36 phage encodes a FMN-containing flavodoxin polypeptide and we report the expression, purification and enzymatic characterization of the recombinant protein. Purified 0305φ8-36 flavodoxin has near-identical spectral properties to control, purified Escherichia coli flavodoxin. Using in vitro assays we show that 0305φ8-36 flavodoxin can be reconstituted with E. coli flavodoxin reductase and support regio- and stereospecific cytochrome P450 CYP170A1 allyl-oxidation of epi-isozizaene to the sesquiterpene antibiotic product albaflavenone, found in the soil bacterium Streptomyces coelicolor. In vivo, 0305φ8-36 flavodoxin is predicted to mediate the 2-electron reduction of the β subunit of phage-encoded ribonucleotide reductase to catalyse the conversion of ribonucleotides to deoxyribonucleotides during viral replication. Our results demonstrate that this phage flavodoxin has the potential to manipulate and drive bacterial P450 cellular metabolism, which may affect both the host biological fitness and the communal microbiome. Such a scenario may also be applicable in other viral-host symbiotic/parasitic relationships.

Pyrethroid insecticide and milkweed cardenolide interactions on detoxification enzyme activity and expression in monarch caterpillars

Declines of the monarch butterfly population have prompted large-scale plantings of milkweed to restore the population. In North America, there are >73 species of milkweed to choose from for these nationwide plantings. However, it is unclear how different milkweed species affect monarch caterpillar physiology, particularly detoxification enzyme activity and gene expression, given the highly variable cardenolide composition across milkweed species. Here, we investigate the effects of a high cardenolide, tropical milkweed species and a low cardenolide, swamp milkweed species on pyrethroid sensitivity as well as detoxification enzyme activity and expression in monarch caterpillars. Caterpillars fed on each species through the fifth-instar stage and were topically treated with bifenthrin after reaching this final-instar stage. Esterase, glutathione S-transferase, and cytochrome P450 monooxygenase activities were quantified as well as the expression of selected esterase, glutathione S-transferase, ABC transporter, and cytochrome P450 monooxygenase transcripts. There were no significant differences in survival 24 h after treatment with bifenthrin. However, bifenthrin significantly increased glutathione S-transferase activity in caterpillars feeding on tropical milkweed and significantly decreased esterase activity in caterpillars feeding on tropical and swamp milkweed. Significant differential expression of ABC transporter, glutathione S-transferase, and esterase genes was observed for caterpillars feeding on tropical and swamp milkweed and not receiving bifenthrin treatment. Furthermore, significant differential expression of glutathione S-transferase and esterase genes was observed for bifenthrin-treated and -untreated caterpillars feeding on tropical milkweed relative to swamp milkweed. These results suggest that feeding on different milkweed species can affect detoxification and development mechanisms with which monarch caterpillars rely on to cope with their environment.