Unveiling the role of bZIP transcription factors CREB and CEBP in detoxification metabolism of Nilaparvata lugens (Stål).

The basic leucine zipper (bZIP) superfamily is a crucial group of xenobiotics in insects. However, little is known about the function of CAAT enhancer binding proteins (CEBP) and cAMP response element binding protein (CREB) in Nilaparvata lugens. In the present study, NlCEBP and NlCREB were cloned and identified. Quantitative polymerase real-time chain reaction (qRT-PCR) analysis showed the expression of NlCEBP and NlCREB was significantly induced after chemical insecticides exposure. Silencing of NlCEBP and NlCREB increased the susceptibility of N. lugens to insecticides, and the detoxification enzyme activities were also significantly decreased. In addition, comparative transcriptome analysis revealed that 174 genes were significantly co-down-regulated after interfering with the two transcription factors. GO analysis showed that co-down-regulated genes are mostly related to energy transport and metabolic functions indicating the potential regulatory role of NlCEBP and NlCREB in detoxification metabolism. Our research shed lights on the functional roles of transcription factors NlCEBP and NlCREB in the detoxification metabolism of N. lugens, providing a theoretical basis for pest management and comprehensive control of this pest and increasing our understanding of insect toxicology.

Microbiome variation correlates with the insecticide susceptibility in different geographic strains of a significant agricultural pest, Nilaparvata lugens.

Microbiome-mediated insecticide resistance is an emerging phenomenon found in insect pests. However, microbiome composition can vary by host genotype and environmental factors, but how these variations may be associated with insecticide resistance phenotype remains unclear. In this study, we compared different field and laboratory strains of the brown planthopper Nilaparvata lugens in their microbiome composition, transcriptome, and insecticide resistance profiles to identify possible patterns of correlation. Our analysis reveals that the abundances of core bacterial symbionts are significantly correlated with the expression of several host detoxifying genes (especially NlCYP6ER1, a key gene previously shown involved in insecticides resistance). The expression levels of these detoxifying genes correlated with N. lugens insecticide susceptibility. Furthermore, we have identified several environmental abiotic factors, including temperature, precipitation, latitude, and longitude, as potential predictors of symbiont abundances associated with expression of key detoxifying genes, and correlated with insecticide susceptibility levels of N. lugens. These findings provide new insights into how microbiome-environment-host interactions may influence insecticide susceptibility, which will be helpful in guiding targeted microbial-based strategies for insecticide resistance management in the field.

Insight into the regulation of the Nrf2 pathway in response to ascovirus infection in Spodoptera exigua.

BACKGROUND: Ascoviruses are a type of entomopathogenic microorganism with high biological pest control potential and are expected to contribute to the natural control of lepidopteran pests. However, knowledge of the molecular mechanism underlying the biocidal activity of ascovirus on its host insects remains limited. RESULTS: In this study, the relative enzyme activity of superoxide dismutase and peroxidase, as well as the expression level of Spodoptera exigua peroxidase (SePOD), were found to be significantly increased at 6 h post infection with Heliothis virescens ascovirus 3h (HvAV-3h). H2 O2 accumulation and enhanced expression of NADPH Oxidase (SeNOX) were also observed. In addition, Nuclear Factor erythroid 2-Related Factor 2 (SeNrf2) and muscle aponeurosis fibromatosis (SeMaf) were overexpressed following infection with HvAV-3h. Silencing of SeNrf2 decreased the expression of SePOD, whereas the mortality of SeNrf2-silenced larvae and viral genome copy number also increased. Further RNA interference of SeNOX significantly decreased expression of SeNrf2 and SePOD and therefore increased the mortality and viral genome copy number of the ascovirus-infected host. CONCLUSION: The HvAV-3h activated Nrf2/ARE pathway of S. exigua and reactive oxygen species were found to respond to ascovirus infection by regulating alterations in antioxidant enzyme genes mediated by the host Nrf2/ARE pathway. These findings enhance our knowledge of ascovirus-host interactions and lay the foundation for the application of ascoviruses in biological pest control. © 2022 Society of Chemical Industry.

Insecticide Susceptibility and Mechanism of Spodoptera frugiperda on Different Host Plants.

Spodoptera frugiperda (J. E. Smith) is a worldwide economically important crop pest. Although the individuals of S. frugiperda that invaded China have been characterized as the corn strain, they also have the ability to damage other crops in China. The physiological and behavioral responses of S. frugiperda to different host plants are poorly understood. In the present study, we investigated the host plant preference, fitness costs, and differences in detoxification gene expression and microbiome composition between two S. frugiperda strains that fed on different crop plant diets. The results showed that S. frugiperda larvae exhibited no obvious preference for corn or rice, but significant suppression of development was observed in the rice-fed strain. In addition, the corn-fed strain showed higher insecticide tolerance and detoxification enzyme activities than the rice-fed strain. Moreover, multiple detoxification genes were upregulated in the corn-fed strain, and microbiome composition variation was observed between the two strains. Together, the results suggest that population-specific plasticity is related to host plant diets in S. frugiperda. These results provide a theoretical basis for the evolution of resistance differences in S. frugiperda and are helpful for designing resistance management strategies for S. frugiperda aimed at different crops.

miRNA novel_268 targeting NlABCG3 is involved in nitenpyram and clothianidin resistance in Nilaparvata lugens.

The brown planthopper (BPH), Nilaparvata lugens (Stål), is one of the most destructive pests that seriously threatens the high-quality and safe production of rice. However, due to the unscientific use of chemical insecticides, N. lugens has developed varying levels of resistance to insecticides, including nitenpyram and clothianidin. The ATP-binding cassette (ABC) transporter plays a nonnegligible role in phase III of the detoxification process, which may play an important role in insecticide resistance. In the present study, NlABCG3 was significantly overexpressed in both the NR and CR populations compared with susceptible populations. Silencing NlABCG3 significantly increased the susceptibility of BPH to nitenpyram and clothianidin. In addition, RNAi-mediated knockdown of three key genes in the miRNA biogenesis pathway altered the level of NlABCG3. Subsequently, the luciferase reporter assays demonstrated that novel_268 binds to the NlABCG3 coding region and downregulates its expression. Furthermore, injection of miRNA inhibitors or mimics of novel_268 significantly altered the susceptibility of N. lugens to nitenpyram and clothianidin. These results suggest that miRNA novel_268 targeting NlABCG3 is involved in nitenpyram and clothianidin resistance in N. lugens. These findings may help to enhance our knowledge of the transcriptional regulation of the ABC transporter that mediate insecticide resistance in N. lugens.

Odorant binding protein 3 is associated with nitenpyram and sulfoxaflor resistance in Nilaparvata lugens.

Odorant binding protein (OBP) can interact with small-molecule compounds insecticides and thereby modulate variation in insecticide susceptibility in insects. However, the regulatory mechanism of OBP-mediated insecticide resistance in Nilaparvata lugens, a destructive rice pest in Asia, remains unclear. Here, we explored the role of NlOBP3 in the resistance of N. lugens to nitenpyram and sulfoxaflor. The results showed that NlOBP3 was overexpressed in association with nitenpyram and sulfoxaflor resistance, and NlOBP3 silencing significantly increased the mortality of N. lugens to nitenpyram and sulfoxaflor, suggesting that NlOBP3 may be associated with nitenpyram and sulfoxaflor resistance in N. lugens. OBP localization revealed that NlOBP3 was highly expressed in all nymph stages and was enriched in the antennae, legs, body wall, and fat body. RT-qPCR analyses showed that the mRNA levels of NlOBP3 were significantly affected by nitenpyram and sulfoxaflor. Additionally, molecular docking predicted that there were multiple binding sites that may played key roles in the binding of NlOBP3 with nitenpyram and sulfoxaflor. The current study identifies a previously undescribed mechanism of insecticide resistance in N. lugens, showing that NlOBP3 is likely to be involved in the evolution of nitenpyram and sulfoxaflor resistance in N. lugens.

miRNAs targeting CYP6ER1 and CarE1 are involved in nitenpyram resistance in Nilaparvata lugens.

The evolution of nitenpyram resistance has been confirmed to be related to overexpression of two key metabolic enzyme genes, CYP6ER1 and CarE1, in Nilaparvata lugens, a highly destructive rice pest that causes substantial economic losses and has developed insecticide resistance. As microRNAs (miRNAs) are important post-transcriptional regulators of gene expression, whether they are involved in nitenpyram resistance is poorly understood in N. lugens. In this study, knockdown of key genes in the miRNA biogenesis pathway (Dicer1, Drosha, and Argonaute1) changed CYP6ER1 and CarE1 abundance, which confirmed the importance of miRNAs in nitenpyram resistance. Furthermore, global screening of miRNAs associated with nitenpyram resistance in N. lugens was performed, and a total of 42 known and 178 novel miRNAs were identified; of these, 57 were differentially expressed between the susceptible and resistant strains, and two (novel_85 and novel_191) were predicted to target CYP6ER1 and CarE1, respectively. Luciferase reporter assays demonstrated that novel_85 and novel_191 bind to the CYP6ER1 and CarE1 coding regions, respectively, and downregulate their expression. Moreover, modulating novel_85 and novel_191 expression by injection of miRNA inhibitors and mimics significantly altered N. lugens nitenpyram susceptibility. This is the first study to systematically screen and identify miRNAs associated with N. lugens nitenpyram resistance, and provides important information that can be used to develop new miRNA-based targets in insecticide resistance management.

Insecticide Resistance Monitoring in Field Populations of the Whitebacked Planthopper Sogatella furcifera (Horvath) in China, 2019-2020.

Monitoring is an important component of insecticide resistance management. In this study, resistance monitoring was conducted on 18 field populations in China. The results showed that S. furcifera developed high levels of resistance to chlorpyrifos and buprofezin, and S. furcifera showed low to moderate levels of resistance to imidacloprid, thiamethoxam, dinotefuran, clothianidin, sulfoxaflor, isoprocarb and ethofenprox. Sogatella furcifera remained susceptible or low levels of resistance to nitenpyram. LC50 values of nitenpyram and dinotefuran, imidacloprid, thiamethoxam, clothianidin and chlorpyrifos exhibited significant correlations, as did those between dinotefuran and thiamethoxam, clothianidin, sulfoxaflor, imidacloprid, isoprocarb and buprofezin. Similarly, significant correlations were observed between thiamethoxam and clothianidin, sulfoxaflor and imidacloprid. In addition, the activity of EST in field populations of S. furcifera were significantly correlated with the LC50 values of nitenpyram, thiamethoxam and clothianidin. These results will help inform effective insecticide resistance management strategies to delay the development of insecticide resistance in S. furcifera.

Resistance of Nilaparvata lugens (Hemiptera: Delphacidae) to triflumezopyrim: inheritance and fitness costs.

BACKGROUND: Triflumezopyrim, a novel commercialized mesoionic chemical insecticide, has been confirmed as a promising insecticide for efficiently controlling the brown planthopper, Nilaparvata lugens (Stål). Here, a laboratory triflumezopyrim-resistant (TR) strain and an isogenic susceptible (TS) strain were established to characterize the inheritance and fitness costs of triflumezopyrim resistance in N. lugens. RESULTS: After 29 generations of successive selection with triflumezopyrim, the TR strain developed a 155.23-fold higher resistance level than the TS strain. The median lethal concentration (LC50 ) values from progenies (F1 RS and F1 SR) of reciprocal crosses between TR and TS strains suggested that triflumezopyrim resistance in N. lugens was autosomal and codominant. Chi-square analyses of self-bred and backcrossed progenies suggested that the resistance results from a polygenic effect. Compared to the TS strain, the TR strain showed a lower relative fitness (0.62) with a significantly decreased female adult period, longevity, total fecundity, egg hatchability, intrinsic rate of increase (r), finite rate of increase (λ), net reproductive rate (R0 ), and prolonged pre-adult period and total preoviposition period (TPOP). CONCLUSION: The inheritance mode of triflumezopyrim resistance in N. lugens was characterized as autosomal, codominant and polygenic. The resistance had a fitness cost, which may be an important factor limiting the evolution of resistance. These findings provide valuable information for optimizing resistance management strategies to delay triflumezopyrim resistance development and maintain sustainable control of N. lugens. © 2021 Society of Chemical Industry.

Functional characterization of the transcription factors AhR and ARNT in Nilaparvata lugens.

In the present study, the aryl hydrocarbon receptor (AhR) and aryl hydrocarbon receptor nuclear translocator (ARNT) of Nilaparvata lugens were cloned and identified. The NlAhR and NlARNT expression levels significantly increased after imidacloprid, etofenprox and isoprocarb treatments. Knockdowns of NlAhR and NlARNT increased the susceptibility of N. lugens to imidacloprid, etofenprox and isoprocarb, and the detoxification enzyme activities were also significantly decreased. In addition, NlCYP301A1, NlGSTt1 and NlCarE7 were significantly down-regulated after injections of dsNlAhR and dsNlARNT, with the NlCarE7 expression decreasing by greater than 80%. Moreover, after knocking down NlCarE7, the susceptibility of N. lugens to etofenprox and isoprocarb significantly increased. Both NlAhR and NlARNT bound the NlCarE7 promoter and significantly enhanced the transcriptional activity. Our research revealed the functional roles of transcription factors NlAhR and NlARNT in the detoxification metabolism of N. lugens. The results provide a theoretical basis for the pest management and comprehensive control of N. lugens and increase our knowledge of insect toxicology.

Dual oxidase-dependent reactive oxygen species are involved in the regulation of UGT overexpression-mediated clothianidin resistance in the brown planthopper, Nilaparvata lugens.

BACKGROUND: Uridine diphosphate-glycosyltransferases (UGTs) are phase II metabolic enzymes involved in metabolism of toxins and resistance to insecticides in insect pests. Reactive oxygen species (ROS) induced by xenobiotics are important for activation of detoxification pathways. However, relationships between ROS and UGTs involved in toxin metabolism and insecticide resistance remain unclear. RESULTS: Here, involvement of dual oxidase (Duox)-dependent ROS in regulating UGT expression-mediated insecticide resistance in the brown planthopper (Nilaparvata lugens) was investigated. The overexpression of NlUGT386F2 contributed to the resistance of N. lugens to clothianidin. Furthermore, the ROS inhibitor (N-acetylcysteine) significantly reduced the expression of NlUGT386F2 and increased the susceptibility of N. lugens to clothianidin. Silencing the ROS producer Duox significantly increased the susceptibility of N. lugens to clothianidin through the down-regulation of NlUGT386F2 expression. CONCLUSION: NlDuox-dependent ROS regulates NlUGT386F2 expression-mediated clothianidin resistance in brown planthopper. These observations further our understanding of the metabolism of toxins and of insecticide-resistance in insect pests.

Activator protein-1 mediated CYP6ER1 overexpression in the clothianidin resistance of Nilaparvata lugens (Stål).

BACKGROUND: Nilaparvata lugens, a destructive rice pest in Asia, has developed resistance to many insecticides, including the neonicotinoid clothianidin. CYP6ER1 plays an important role in N. lugens resistant to clothianidin, but only limited information on the transcriptional regulation of CYP6ER1 overexpression in clothianidin resistance is available. RESULTS: In this study, the transcription factor activator protein 1 (AP-1) was found to be overexpressed in a clothianidin-resistant strain of N. lugens and several field resistant populations. RNA interference-mediated silencing of NlAP-1 significantly decreased CYP6ER1 expression and increased the susceptibility of N. lugens to clothianidin. Additionally, NlAP-1 was highly expressed in egg and adult stages, and in midguts, and NlAP-1 was upregulated and induced to a greater extent in the clothianidin-resistant strain after exposure to clothianidin. Finally, dual-luciferase reporter assays confirmed the interaction between NlAP-1 and the two predicted binding sites in the CYP6ER1 promoter. CONCLUSION: NlAP-1 bound the -1388 to -1208-bp region of the CYP6ER1 promoter, enhancing its activity and then regulate the expression of CYP6ER1. These findings enhance our knowledge of the transcriptional regulation of the P450 genes that mediate insecticide resistance in insect pests. © 2021 Society of Chemical Industry.

Transcriptional Response of ATP-Binding Cassette (ABC) Transporters to Insecticide in the Brown Planthopper, Nilaparvata lugens (Stål).

The ATP-binding cassette (ABC) transporter superfamily is one of the largest groups of proteins and plays a non-negligible role in phase III of the detoxification process, which is highly involved in the response of insects to environmental stress (plant secondary metabolites and insecticides). In the present study, in Nilaparvata lugens, we identified 32 ABC transporters, which are grouped into eight subfamilies (ABCA-H) based on phylogenetic analysis. The temporal and spatial expression profiles suggested that the nymphal stages (1st-5th) and adult males showed similarity, which was different from eggs and adult females, and NlABCA1, NlABCA2, NlABCB6, NlABCD2, NlABCG4, NlABCG12, NlABCG15, and NlABCH1 were highly expressed in the midgut and Malpighian tubules. In addition, ABCG12, which belongs to the ABC transporter G subfamily, was significantly upregulated after exposure to sulfoxaflor, nitenpyram, clothianidin, etofenprox, chlorpyrifos, and isoprocarb. Moreover, verapamil significantly increased the sensitivity of N. lugens to nitenpyram, clothianidin, etofenprox, chlorpyrifos, and isoprocarb. These results provide a basis for further research on ABC transporters involved in detoxification in N. lugens, and for a more comprehensive understanding of the response of N. lugens to environmental stress.

Cross-resistance and biochemical characterization of buprofezin resistance in the white-backed planthopper, Sogatella furcifera (Horvath).

Buprofezin is a chitin synthesis inhibitor that is very effective against Homopteran pests, such as the white-backed planthopper (WBPH), S. furcifera (Horvath). In the present study, resistance selection, cross-resistance and mechanisms of buprofezin resistance were investigated in this planthopper species. However, the mechanism associated with resistance to growth regulator insecticides (IGRs) remains largely unknown. A resistant strain (Bup-R) with a resistance level (22-fold) to buprofezin was developed through continuous selection for 47 generations from a laboratory susceptible strain (Bup-S). The results showed that the Bup-R exhibited no cross-resistance to other tested insecticides. Synergism tests showed that piperonyl butoxide (PBO) (SR = 3.9-fold) and diethyl maleate (DEM) (SR = 1.8-fold) had synergistic effects on buprofezin toxicity in the resistant strain (F47). Enzyme activity results revealed an approximate 5.7-fold difference in cytochrome P450 monooxygenase and a 2-fold difference in glutathione S-transferase (GST) between the resistant and susceptible strains, suggesting that the increased activity of these two enzymes is likely the main detoxification mechanism involved in resistance to buprofezin in this species. Furthermore, the mRNA expression levels of cytochrome P450 (CYP) and GST genes by quantitative real-time PCR results indicated that sixteen P450 and one GST gene were significantly overexpressed in the Bup-R strain, among which thirteen P450 genes and one GST gene were >2-fold higher than in the Bup-S strain. The present study increases our knowledge of the buprofezin resistance mechanism in S. furcifera and provides a useful reference for integrated pest management (IPM) strategies.

Characterization of nitenpyram resistance in Nilaparvata lugens (Stål).

Nitenpyram is very effective in controlling Nilaparvata lugens (brown planthopper, BPH), and its resistance has been reported in field populations; however, the resistance mechanism remains unclear. In the present study, cross-resistance and resistance mechanisms in nitenpyram-resistant BPH were investigated. A resistant strain (NR) with a high resistance level (164.18-fold) to nitenpyram was evolved through successive selection for 42 generations from a laboratory susceptible strain (NS). The bioassay results showed that the NR exhibited cross-resistance to imidacloprid (37.46-fold), thiamethoxam (71.66-fold), clothianidin (149.17-fold), dinotefuran (98.13-fold), sulfoxaflor (47.24-fold), cycloxaprid (9.33-fold), etofenprox (10.51-fold) and isoprocarb (9.97-fold) but not to triflumezopyrim, chlorpyrifos and buprofezin. The NR showed a 3.21-fold increase in cytochrome P450 monooxygenase (P450) activity compared to that in the NS, while resistance was also synergized (4.03-fold) with the inhibitor piperonyl butoxide (PBO), suggesting a role of P450. Furthermore, the mRNA expression levels of cytochrome P450 (CYP) genes by quantitative real-time PCR results indicated that twelve P450 genes were significantly overexpressed in the NR strain, especially CYP6ER1 (203.22-fold). RNA interference (RNAi) suppression of CYP6ER1 through injection of dsCYP6ER1 led to significant susceptibility in the NR strain. The current study expands our understanding of the nitenpyram resistance mechanism in N. lugens, provides an important reference for integrated pest management (IPM), and enriches the theoretical system of insect toxicology.

The influence of temperature on the toxicity of insecticides to Nilaparvata lugens (Stål).

The toxicity of insecticides is associated with a variety of factors including temperature, and global warming is bound to lead to the outbreak of pests; therefore, it is important to study the influence of temperature on insecticide toxicity and pest control. In this study, the influence of temperature on the toxicity of insecticides to Nilaparvata lugens (BPH) was determined. The results showed that the sensitivity of BPH to cycloxaprid (LC50 = 42.5-0.388 mg/L), nitenpyram (LC50 = 3.49-0.187 mg/L), triflumezopyrim (LC50 = 0.354-0.0533 mg/L) and chlorpyrifos (LC50 = 36.3-7.41 mg/L) increased significantly when the temperature changed from 18 °C to 36 °C. BPH sensitivity to etofenprox (LC50 = 9.04-54.2 mg/L) was also affected by temperature. Additionally, the feeding amount and the activities of three detoxification enzymes [cytochrome P450 (P450), glutathione S-transferase (GST) and carboxylesterase (CarE)] of BPH at different temperatures were also measured. The feeding amounts were positively correlated with temperature increases while the activities of P450 and GST were significantly inhibited. The correlation analysis showed that changes in P450 activity (but not GST activity) were closely related to the sensitivity of BPH to cycloxaprid, nitenpyram, chlorpyrifos, and etofenprox according to the variation in temperatures. This study provides a theoretical basis for the rational use of chemical pesticides under the global warming trend and provides a reference for the integrated management of BPH in the field.

Inheritance and fitness costs of sulfoxaflor resistance in Nilaparvata lugens (Stål).

BACKGROUND: Sulfoxaflor has been considered as a new tool for Nilaparvata lugens control in the field. In this study, a laboratory-selected resistant strain and a susceptible strain were used to evaluate the inheritance and fitness costs of sulfoxaflor resistance in N. lugens. RESULTS: The resistant strain (SFX-SEL) showed 123.63-fold resistance compared with the susceptible strain (SS). Progenies of reciprocal crosses (F1 RS and F1 SR) showed similar concentration-mortality responses (LC50 ) to sulfoxaflor and also exhibited a similar degree of dominance; -0.16 for F1 RS and -0.26 for F1 SR. Significant differences between the observed and expected mortalities of F2 individuals suggested that sulfoxaflor resistance is associated with multiple genes. The resistant strain had a relative fitness of 0.75 with substantially decreased female adult period, oviposition days, total fecundity, egg hatchability and female adult survival rate, and prolonged pre-adult period and total pre-oviposition period. CONCLUSION: Sulfoxaflor resistance in N. lugens was inherited as autosomal, incompletely recessive and multigene. The development of resistance may have a significant fitness cost for the resistant population. Current research provides valuable information for researchers to establish management strategies to delay the development of sulfoxaflor resistance and control N. lugens sustainably in the field. © 2019 Society of Chemical Industry.

Overexpression of CYP6ER1 associated with clothianidin resistance in Nilaparvata lugens (Stål).

The brown planthopper, Nilaparvata lugens (Stål), is one of the most economically important rice pests in Asia and has become resistant to various kinds of insecticides, including neonicotinoid insecticides. In this study, an N. lugens clothianidin-resistant (CLR) strain and a susceptible (CLS) strain were established, and the potential resistance mechanisms of N. lugens to clothianidin were elucidated. The cross-resistance studies showed that the clothianidin-resistant strain exhibited cross-resistance to most neonicotinoid insecticides, especially nitenpyram (99.19-fold) and dinotefuran (77.68-fold), while there was no cross-resistance to chlorpyrifos (1.79-fold). The synergism assays and the activities of the detoxification enzymes were performed, and we found that a cytochrome P450 conferred the clothianidin resistance. Two P450 genes (CYP6ER1 and CYP6AY1) were found to be significantly overexpressed in the CLR strain compared with the CLS strain based on qRT-PCR. In addition, the knockdown of CYP6ER1 by RNA interference dramatically increased the toxicity of clothianidin against N. lugens. These data demonstrated that the overexpression of CYP6ER1 could contribute to clothianidin resistance in N. lugens. Our findings will help to improve the design of effective resistance management strategies to control brown planthoppers.

Characterization of sulfoxaflor resistance in the brown planthopper, Nilaparvata lugens (Stål).

BACKGROUND: Sulfoxaflor is a new insecticide for controlling Nilaparvata lugens in the field. This study was conducted to investigate the risk of resistance development, the cross-resistance spectrum and the mechanisms of sulfoxaflor resistance in N. lugens. RESULTS: A sulfoxaflor-resistant strain was obtained from a field population by successive selection with sulfoxaflor for 39 generations in the laboratory. Sulfoxaflor-resistant populations showed significant levels of cross-resistance to dinotefuran, nitenpyram, thiamethoxam, clothianidin, imidacloprid and cycloxaprid. However, they exhibited only minor or no cross-resistance to isoprocarb, etofenprox, chlorpyrifos, triflumezopyrim and buprofezin. Sulfoxaflor was synergized by the inhibitor piperonyl butoxide (PBO) in the sulfoxaflor-resistant strain (SFX-SEL) with 2.69-fold relative synergistic ratios compared with the unselected strain (UNSEL). Compared with UNSEL, the P450 enzyme activity of SFX-SEL was increased 3.50 times, and eight P450 genes were upregulated more than 2.0-fold in SFX-SEL. RNAi reduced the expression of CYP6ER1 (36.87-fold change) and significantly enhanced the susceptibility of SFX-SEL to sulfoxaflor. CONCLUSION: Resistance development and cross-resistance risk of sulfoxaflor-resistance in N. lugens is evident. The enhanced detoxification of P450 enzymes caused by upregulation of several P450 genes is considered to be the metabolic resistance mechanism. These results suggest that CYP6ER1 might play an important role in sulfoxaflor resistance in N. lugens. © 2018 Society of Chemical Industry.

Functional characterization of the aspartic proteinase cathepsin D in the beet armyworm (Spodoptera exigua).

In insects, proteolytic enzymes are involved in food digestion and the metamorphosis process. In the present study, the full-length cDNA of an aspartic proteinase, Spodoptera exigua cathepsin D (SeCatD), was cloned, and its functions in metamorphosis were characterized. SeCatD contains an open reading frame of 1152 nucleotides, encoding a 384-amino acid polypeptide including a signal peptide and two functional domains (family A1 propeptide of amino acids (19-45) and a cathepsin D-like domain of 327 amino acids (55-381)). Three-dimensional structure analysis indicated that Asp66 and Asp251 may play important role in hydrolysis. Recombinant SeCatD was expressed in Sf9 insect cells and verified via SDS-PAGE and Western blot, the molecular mass of the expressed SeCatD was approximately 42kDa. The enzyme had an optimal pH value of 3 for activity. In addition, the tissue expression profile of SeCatD during metamorphosis was obtained, and the data demonstrated that SeCatD was expressed increasingly in the fat body and midgut, but not in the epidermis. Finally, injection of dsRNA-SeCatD into the fifth-instar larvae significantly reduced SeCatD expression and larvae survival rate compared to a dsRNA-GFP treatment. These data imply that SeCatD may function during metamorphosis and may represent a target for insect control.