Enhancement of tolerance against flonicamid in Solenopsis invicta (Hymenoptera: Formicidae) through overexpression of CYP6A14.

Solenopsis invicta is a main issue in southern China and is causing significant damage to the local ecological environment. The extensive use of insecticides has resulted in the development of tolerance in S. invicta. In our study, ten S. invicta colonies from Sichuan Province exhibited varying degrees of tolerance against flonicamid, with LC50 values from 0.49 mg/L to 8.54 mg/L. The sensitivity of S. invicta to flonicamid significantly increased after treatment with the P450 enzyme inhibitor piperonyl butoxide (PBO). Additionally, the activity of P450 in S. invicta was significantly enhanced after being treated with flonicamid. Flonicamid induced the expression levels of CYP4aa1, CYP9e2, CYP4C1, and CYP6A14. The expression levels of these P450 genes were significantly higher in the tolerant colonies compared to the sensitive colonies, and the relative copy numbers of CYP6A14 in the tolerant colonies were 2.01-2.15 fold. RNAi feeding treatment effectively inhibited the expression of P450 genes, thereby reducing the tolerance of S. invicta against flonicamid. In addition, the overexpression of CYP6A14 in D. melanogaster resulted in reduced sensitivity to flonicamid. Our investigations revealed hydrophobic interactions between flonicamid and seven amino acid residues of CYP6A14, along with the formation of a hydrogen bond between Glu306 and flonicamid. Our findings suggest that flonicamid can effectively control S. invicta and P450 plays a pivotal role in the tolerance of S. invicta against flonicamid. The overexpression of CYP6A14 also increased tolerance to flonicamid.

Resistance monitoring and mechanism in the fall armyworm Spodoptera frugiperda (Lepidoptera: Noctuidae) for chlorantraniliprole from Sichuan Province, China.

The fall armyworm, Spodoptera frugiperda (Noctuidae: Lepidoptera), is a wide-reaching notorious insect pest of important cereal crops, which has developed resistance to multiple classes of insecticides. It invaded the Sichuan Province of China in 2019. In this study, we performed resistance monitoring of insecticides for 11 field-collected populations from Sichuan, and all the populations were susceptible to emamectin benzoate and chlorpyrifos. The variations in resistance level to indoxacarb (resistance ratio (RR), 9.23-45.53-fold), spinetoram (RR, 4.32-18.05-fold), and chlorantraniliprole (RR, 2.02-10.39-fold) were observed among these populations. To investigate the resistance mechanism of chlorantraniliprole, synergism tests were performed and showed that piperonyl butoxide had a slight synergistic effect on chlorantraniliprole for the QJ-20 population (1.43-fold) in moderate resistance (RR, 10.39-fold) compared with the treatment group without synergist. Furthermore, the expression scanning for resistance-related genes showed that five P450 genes (CYP6AE43, CYP321A8, CYP305A1, CYP49A1, and CYP306A1) and the ryanodine receptor gene (Ryr, chlorantraniliprole target) were overexpressed in the QJ-20 population. These results indicated that the fall armyworm in Sichuan has exhibited diverse susceptibilities to several classes of insecticides, and the overexpression of Ryr and several P450 genes may contribute to the development of resistance in S. frugiperda to chlorantraniliprole.

Differences in susceptibility to chlorantraniliprole between Chilo suppressalis (Lepidoptera: Crambidae) and two dominant parasitic wasps collected from Sichuan Province, China.

Chilo suppressalis Walker (Lepidoptera: Crambidae) is one of the most destructive pests occurring in the rice-growing regions of Asia. Parasitoids, mainly egg parasitoids, have been of interest for several years even with practical used cases. Therefore, the potential impact of insecticides on natural enemies needs great attention. In this study, chlorantraniliprole was evaluated for its impact on C. suppressalis and two dominant parasitic wasps. Bioassays showed that chlorantraniliprole had negligible toxicity to Eriborus terebrans but was significantly toxic to Chelonus munakatae; the mortality exceeded 50% when the concentration reached 46.83 ng/cm2. Enzyme assays suggested that the significantly different carboxylesterase activity may be involved in the high-level detoxification metabolism of E. terebrans. According to the results of enzyme gene correlation analysis, P450s may be the dominant factor in the detoxification metabolism of C. munakatae. In addition, the ryanodine receptor C-terminus of C. suppressalis (CsRyR), C. munakatae (CmRyR) and E. terebrans (EtRyR) were successfully cloned. Different amino acids at resistance mutation I4758 M between susceptible C. suppressalis (I) and parasitic wasps (M) may be related to susceptibility differences. Simulated docking showed that CsRyR and CmRyR can interact with chlorantraniliprole but not EtRyR. More interaction forces were formed between CsRyR and chlorantraniliprole than CmRyR. Furthermore, a Pi-Pi T-shape formed between 73PHE in CsRyR and the benzene ring in chlorantraniliprole. These results indicated that both detoxification metabolism and the target site could mediate the susceptibility difference between C. suppressalis and its parasitic wasps.

Mechanisms for multiple resistances in field populations of rice stem borer, Chilo suppressalis (Lepidoptera: Crambidae) from Sichuan Province, China.

Chilo suppressalis Walker (Lepidoptera: Crambidae) is a widely destructive pest occurring in rice, particularly in the rice-growing regions of Asia. In recent years, C. suppressalis has developed resistance to several insecticides because of the extensive use of insecticides. The resistance levels to four insecticides were determined among populations from different regions of Sichuan Province, China, using a drop-method bioassay. Based on LC50 values of a laboratory susceptible strain, all field populations showed moderate level of resistance to triazophos (23.9- to 83.5-fold) and were either susceptible or had a low level of resistance to abamectin (2.1- to 5.8-fold). All field-collected populations had a low or moderate level of resistance to chlorpyrifos (1.7- to 47.1-fold) and monosultap (2.7- to 13.5-fold). The synergism experiment indicated that the resistance of the XW19 to triazophos may be associated with cytochrome P450 monooxygenases (P450s), with the highest synergistic ratio (SR) of 3.05-fold and increased ratio (IR) of 2.28-fold for piperonylbutoxide (PBO). The P450 activity of the TJ19 population was the greatest among the six field populations. Moreover, the relative expression levels of four resistance-related P450 genes were detected with qRT-PCR, and the results indicated that CYP324A12, CYP321F3 and CYP9A68 were overexpressed in the resistant population, especially in the XW19 population (by 1.2-, 3.4 -, and 18.0-fold, respectively). In addition, the relative expression levels of CYP9A68 among the CZ19 and TJ19 populations were also enhanced 10.5- and 24.9-fold, respectively. These results suggested that CYP324A12, CYP321F3 and CYP9A68 may be related to the resistance development of C. suppressalis to triazophos.

Transcriptome Analysis of Sogatella furcifera (Homoptera: Delphacidae) in Response to Sulfoxaflor and Functional Verification of Resistance-Related P450 Genes.

The white-back planthopper (WBPH), Sogatella furcifera, is a major rice pest in China and in some other rice-growing countries of Asia. The extensive use of pesticides has resulted in severe resistance of S. furcifera to variety of chemical insecticides. Sulfoxaflor is a new diamide insecticide that acts on nicotinic acetylcholine receptors (nAChRs) in insects. The aim of this study was to explore the key genes related to the development of resistance to sulfoxaflor in S. furcifera and to verify their functions. Transcriptomes were compared between white-back planthoppers from a susceptible laboratory strain (Sus-Lab) and Sus-Lab screened with the sublethal LC25 dose of sulfoxaflor for six generations (SF-Sel). Two P450 genes (CYP6FD1 and CYP4FD2) and three transcription factors (NlE78sf, C2H2ZF1 and C2H2ZF3) with upregulated expression verified by qRT-PCR were detected in the Sus-Lab and SF-Sel strains. The functions of CYP6FD1 and CYP4FD2 were analyzed by RNA interference, and the relative normalized expressions of CYP6FD1 and CYP4FD2 in the SF-Sel population were lower than under dsGFP treatment after dsRNA injection. Moreover, the mortality rates of SF-Sel population treated with the LC50 concentration of sulfoxaflor after the injecting of dsRNA targeting CYP6FD1 and CYP4FD2 were significantly higher than in the dsGFP group from 72 h to 96 h (p < 0.05), and mortality in the CYP6FD1 knockdown group was clearly higher than that of the CYP4FD2 knockdown group. The interaction between the tertiary structures of CYP6FD1 and CYP4FD2 and sulfoxaflor was also predicted, and CYP6FD1 showed a stronger metabolic ability to process sulfoxaflor. Therefore, overexpression of CYP6FD1 and CYP4FD2 may be one of the primary factors in the development of sulfoxaflor resistance in S. furcifera.