Soil cadmium pollution facilitated the invasion of alligator weed through enhanced herbivore resistance and competitive ability over a congeneric species.

A number of invasive plant species, such as Alternanthera philoxeroides, have been documented to be able to accumulate trace metal elements in their tissues. Since metal accumulation in plants can serve as a defence against herbivores, we hypothesized that metal pollution will increase herbivore resistance of metal-accumulating invasive plant species and such a benefit will grant them a competitive advantage over local co-occurring plants. In this study, we compared the differences in plant growth and herbivore feeding preference between A. philoxeroides and its native congener Alternanthera sessilis in single and mixed cultures with and without soil cadmium (Cd) pollution. The results showed that A. philoxeroides plants were more tolerant to Cd stress and accumulated more Cd in the leaves than A. sessilis. Cd exposure increased the resistance of A. philoxeroides against a specialist and a generalist herbivore compared with A. sessilis. Competition experiments indicated that Cd stress largely increased the competitive advantage of A. philoxeroides over A. sessilis with or without herbivore pressures. The differences in herbivore resistance between the two plant species under soil Cd stress are most likely due to the deterring effect of Cd accumulation and Cd-enhanced mechanical defences rather than changes in leaf specialized metabolites.

Sphingomonas bacteria could serve as an early bioindicator for the development of chlorantraniliprole resistance in Spodoptera frugiperda.

The fall armyworm (Spodoptera frugiperda) was found to have invaded China in December 2018, and in just one year, crops in 26 provinces were heavily affected. Currently, the most effective method for emergency control of fulminant pests is to use of chemical pesticides. Recently, most fall armyworm populations in China were begining to exhibite low level resistance to chlorantraniliprole. At present, it is not possible to sensitively reflect the low level resistance of S. frugiperda by detecting target mutation and detoxification enzyme activity. In this study we found that 12 successive generations of screening with chlorantraniliprole caused S. frugiperda to develop low level resistance to this insecticide, and this phenotype was not attribute to genetic mutations in S. frugiperda, but rather to a marked increase in the relative amount of the symbiotic bacteria Sphingomonas. Using FISH and qPCR assays, we determined the amount of Sphingomonas in the gut of S. frugiperda and found Sphingomonas accumulation to be highest in the 3rd-instar larvae. Additionally, Sphingomonas was observed to provide a protective effect to against chlorantraniliprole stress to S. frugiperda. With the increase of the resistance to chlorantraniliprole, the abundance of bacteria also increased, we propose Sphingomonas monitoring could be adapted into an early warning index for the development of chlorantraniliprole resistance in S. frugiperda populations, such that timely measures can be taken to delay or prevent the widespread propagation of resistance to this highly useful agricultural chemical in S. frugiperda field populations.

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.

Status of Fungicide Resistance and Physiological Characterization of Tebuconazole Resistance in Rhizocotonia solani in Sichuan Province, China.

The resistance prevalence of chemical fungicides has caused increasingly serious agro-ecological environmental problems. However, there are few previous reports about resistance to succinate dehydrogenase (SDHI) or sterol demethylation inhibitor (DMI) in Rhizoctonia solani, one of the main agro-diseases. In this study, the fungicide resistance of 122 R. solani isolates in Sichuan Province was monitored by the mycelial growth rate method. Results showed that all isolates were susceptible to hexaconazole and most isolates were susceptible to thifluzamide, except for the field isolate MSRS-2-7 due to a moderate resistance to thifluzamide (16.43-fold resistance ratio, RR), compared to the sensitivity baseline of thifluzamide (0.042 µg/mL EC50 values). On the contrary, many isolates showed moderate or high resistance to tebuconazole (10.59- to 60.78-fold RR), reaching EC50 values of 0.54~3.10 µg/mL, especially for a highly resistant isolate LZHJ-1-8 displaying moderate resistance to epoxiconazole (35.40-fold RR due to a 3.54 µg/mL EC50 value). The fitness determination found that the tebuconazole-resistant isolates showed higher fitness cost with these characteristics, including a lower growth rate, higher relative electric conductivity, an increased ability to tolerate tebuconazole, and high osmotic pressure. Four new mutations of cytochrome P450 sterol 14α-demethylase (CYP51), namely, S94A, N406S, H793R, and L750P, which is the target for DMI fungicides, was found in the tebuconazole-resistant isolates. Furthermore, the lowest binding energy with tebuconazole was also found in the LZHJ-1-8 isolate possessing all the mutations through analyses with Discovery Studio software. Therefore, these new mutation sites of CYP51 may be linked to the resistance against tebuconazole, and its application for controlling R. solani should be restricted in some areas.

Resistance of Sogatella furcifera to triflumezopyrim mediated with the overexpression of CYPSF01 which was regulated by nuclear receptor USP.

Sogatella furcifera is one of the main agricultural pests in many Asian countries, bringing about enormous injury. A triflumezopyrim-resistant (Tri) strain of S. furcifera was established through continuous screening in laboratory. The determination of synergist and enzyme activity indicated that P450s, especially for the upregulation expression of CYPSF01, played a key role in the increased resistance, confirmed by RNAi, and the recombinant protein of CYPSF01 and NADPH-P450 reductase was able to degrade triflumezopyrim. CYPSF01 had an obviously co-expression relationship with nuclear receptor ultraspiracle (USP), which were all significantly up-regulated when exposed to triflumezopyrim. Further, a USP-binding motif MA0534.1 was enriched from the upregulated peaks by Assay for Transposase Accessible Chromatin (ATAC-seq) analysis, which exited in the peaks located on the promoter of CYPSF01; the yeast one-hybrid experiments confirmed that USP could bind to the CYPSF01 promoter. And the USP interference significantly down-regulated CYPSF01 expression, and resulted in the significantly increasing sensitivity to triflumezopyrim, its mortality rate increased 28.37%. Therefore, the overexpression of USP could cause to the overexpression of CYPSF01, ultimately resulting in the resistance to triflumezopyrim in S. furcifera.

Maf regulates the overexpression of CYP307A1, which is involved in the fitness advantage of bistrifluron-resistant Spodoptera litura (Fab.) (Noctuidae: Lepidoptera).

Spodoptera litura is a widely distributed multifeeding pest, that has developed high resistance to many chemical insecticides. In the present study, a bistrifluron-resistant (Bis-SEL) strain showing 113.8-fold resistance ratio relative to a bistrifluron-susceptible (Bis-UNSEL) strain was obtained and showed a fitness advantage (resurgence). First, we found that the observed resurgence might have resulted from Maf transcription factor overexpression in the Bis-SEL strain, which would influence the synthesis of ecdysone and chitin. Additionally, a co-expression relationship between Maf and CYP307A1 was verified by weighted correlation network analysis (WGCNA) and qRT-PCR, and the expression of CYP307A1, a key gene in ecdysone synthesis, was significantly downregulated by Maf interference. The assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) and a yeast one-hybrid showed that Maf could bind to the cncc:maf-s element in the CYP307A1 promoter region. The synthesis of ecdysone, which stimulated chitin synthesis, was also decreased significantly following Maf and CYP307A1 interference. Therefore, the upregulation of Maf expression leaded to the upregulation CYP307A1 expression, which led to an increase in the synthesis of ecdysone, resulting in resurgence accompanied by resistance to bistrifluron.

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.

Reproductive Outbreaks of Sogatella furcifera Mediated by Overexpression of the Nuclear Receptor USP under Pressure from Triflumezopyrim.

Long-term pesticide-driven selection pressure is one of the main causes of insect outbreaks. In this study, we found that low doses of triflumezopyrim could increase the fecundity of white-backed planthoppers (Sogatella furcifera). By continuously screening 20 generations with a low dose of triflumezopyrim, a triflumezopyrim-resistant strain (Tri-strain, resistance ratio = 20.9-fold) was obtained. The average oviposition quantity and longevity of the Tri-strain (208.77 eggs and 21.31 days, respectively) were significantly higher than those of the susceptible strain (Sus-strain) (164.62 eggs and 17.85 days, respectively). To better understand the mechanism underlying the effects on reproduction, we detected the expression levels of several reproduction-related transcription factors in both the Tri- and Sus-strains. Ultraspiracle (USP) was significantly overexpressed in the Tri-strain. Knockdown of USP by RNAi severely inhibited the moulting process of S. furcifera and disrupted the development of female adult ovaries. Among the potential downstream target genes of USP, Kr-h1 (0.19-fold), Cht8 (0.56-fold) and GPCR A22 (0.31-fold) showed downregulated expression after USP-RNAi. In contrast, the expression of EcR (2.55-fold), which forms heterodimers with USP, was significantly upregulated. Furthermore, RNAi was performed on Kr-h1 in the Tri-strain, and the results show that larval moulting and the development of female adult ovaries were inhibited, consistent with the USP-RNAi results in S. furcifera. These results suggest that the transcription factors USP and Kr-h1 play important roles in the reproductive development of S. furcifera, and overexpression of USP and Kr-h1 in the Tri-resistant strain may result in reproductive outbreaks of pests.

Exogenous nitrogen enhances poplar resistance to leaf herbivory and pathogen infection after exposure to soil cadmium stress.

Elemental defense hypothesis suggests that toxic metals accumulated in plant tissues could enhance plant defense against herbivores and pathogens. Since over-accumulation of metals in plant organs will pose negative effects on plant health, it is necessary to find a way to alleviate metal-induced toxicity in plants while keeping or even improving plant resistance. Exogenous nitrogen (N) application was reported to have such alleviation effect while stimulating metal accumulation in plant tissues. In this study, we examined whether soil N addition in three different doses to a poplar species under cadmium (Cd) stress can simultaneously improve plant growth and resistance to four herbivorous insects and a leaf pathogen. The results showed that N application to Cd-amended soil prominently enhanced plant growth and leaf Cd accumulation. While N addition in three doses all remarkably reduced herbivore growth than control plants, only the highest N dose exerted stronger inhibition than the sole Cd-treated plants. In the paired-choice experiment, plants supplied with the highest N dose showed an enhanced deterrent effect on herbivore preference than plants exposed to sole Cd. Furthermore, plant resistance to the leaf pathogen infection was strongly enhanced as the levels of N addition increased. Leaf sugar and three main defensive chemicals were not affected by N application implied that such enhanced effect of N on plant resistance was due to increased leaf Cd accumulation. Our results suggested that the application of exogenous N over a certain amount could enhance the resistance of Cd-treated plants to leaf herbivory and pathogen infection.

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.

Status of insecticide resistance and biochemical characterization of chlorpyrifos resistance in Sogatella furcifera (Hemiptera:Delphacidae) in Sichuan Province, China.

The white-backed planthopper, Sogatella furcifera (Horváth) (Hemiptera, Delphacidae), is an energetic rice insect pest in rice production or rice-growing areas. Due to excessive use of the chemical insecticide, S. furcifera has produced the high resistance to some frequently used insecticides. In this paper, the resistance levels of S. furcifera from the eight different areas of Sichuan Province against the five chemicals were monitored by using the rice seedling dipping during 2017-2018 to understand the resistance levels. The results showed that most of all populations have developed low or moderate level of resistance for chlorpyrifos (3.4 to 44.3-fold) and thiamethoxam (3.9- to 15.5-fold), the populations in the LS (1.7 to 5.4- fold)and WS (1.6 to 5.0- fold) regions were still sensitive or low resistance levels compared with other local populations. Almost all populations displayed the susceptible to imidacloprid (0.9- to 5.0-fold), buprofezin (0.9- to 4.3-fold) or low levels of resistance to pymetrozine (1.5- to 6.8-fold). The synergism experiment indicated that P450 enzymes may be important contributed to the metabolic detoxification of chlorpyrifos. The cross-resistance bioassay showed that there was no cross-resistance between chlorpyrifos and triflumezopyrim, but for sulfoxaflor, in the XY17 population. The relative expression level of twelve insecticide resistant-related P450 genes were analyzed by using qRT-PCR and found that CYP4C77, CYP418A1, CYP418A2, CYP408A3 and CYP6ER4 were significantly more expressed in the 3rd-instar nymph of the XY17 and XY18 field populations. To determine the main resistant-related P450 gene for chlorpyrifos, the relative expression level of five P450 genes were detected by using qRT-PCR from the G2 and G4 generation of XY17 under the pressure with LC50 of chlorpyrifos. The results showed that CYP6ER4 was significantly up-regulated expression in XY17 G2 and G4 generations population over 700-fold (P < 0.01). The full length and proteins tertiary structure were also cloned and predicted. Meanwhile, the function of CYP6ER4 was analyzed by RNA interference and the results indicated that the relative expression of CYP6ER4 in the XY17 (G4) population after injected dsRNA was lower than that in the dsGFP injected group. Moreover, the mortality rates of the S. furcifera treated with the LC50 concentration of chlorpyrifos after dsRNA microinjection was significantly higher than that of the dsGFP injected group 72 h after treatment (P < 0.01). Therefore, the overexpression of CYP6ER4 may be one of the primary factors in the development of chlorpyrifos resistance in S. furcifera.

Fitness Costs of Chlorantraniliprole Resistance Related to the SeNPF Overexpression in the Spodoptera exigua (Lepidoptera: Noctuidae).

Spodopteraexigua, a multifeeding insect pest, has developed a high level of resistance to chlorantraniliprole, which is a benzoylurea insecticide that targets the ryanodine receptors (RyRs). Herein, the resistant strain (SE-Sel) and sensitive strain (SE-Sus) were obtained by bidirectional screening for six generations. The potential oviposited eggs and oviposition rate of the SE-Sel strain were dramatically lower than those of the SE-Sus strain; on the contrary, the weights of prepupae and preadult were significantly increased. As a post-mating response, the higher number of non-oviposited eggs in the SE-Sel strain was caused by a lower mating rate. In addition, the expression levels of vitellogenin (SeVg) and its receptor (SeVgR) in the SE-Sel strain were consistently lower than those in the SE-Sus strain. An RyRI4743M mutation, contributing to the resistance to chlorantraniliprole, was located in the S3 transmembrane segments and might have affected the release of calcium ions; it led to the upregulated expression of the neuropeptide SeNPF and its receptor SeNPFR, and the mating and oviposition rate were significantly recovered when the SeNPF was knocked down though RNA interference (RNAi) in the male adult of the SE-Sel strain. Moreover, the expression of the juvenile hormone-binding proteins SeJHBWDS3 and SeJHBAN in the male adult of the SE-Sel strain was significantly decreased, which proved the existence of a fitness cost from another angle. Therefore, these results indicate that the fitness cost accompanied by chlorantraniliprole resistance in S. exigua may be related to the decrease in mating desire due to SeNPF overexpression.

Effects of exogenous spermidine on poplar resistance to leaf and root herbivory as affected by soil cadmium stress.

Elemental defense hypothesis (EDH) proposed that metal accumulation in plants could increase plant resistance to herbivores. However, the over-accumulation of metals in low-accumulators such as woody plants will cause adverse effects on plant health. Thus, the application of EDH on low-accumulators in metal contaminated areas is strongly limited. Since the supplement of exogenous spermidine (Spd) was reported to alleviate metal-induced toxicity in plants while stimulating metal accumulation in plant tissues, we hypothesized that such application will further improve plant resistance to herbivores. In this study, we employed a woody plant species, Populus yunnanensis, to test this hypothesis. We first tested a Spd concentration series applied on plants subjected to soil cadmium (Cd) stress and found that the 1 mM Spd strongly promoted plant growth while stimulated Cd accumulation in plant leaves and roots. We further conducted herbivore bioassays to test the growth performance and feeding preference of two leaf herbivore species and a root herbivore species that fed on plants from different treatments. The results showed that the inhibition effect of Cd stressed-plants on herbivore growth was significantly magnified by the addition of Spd. The growth weight of all the three tested herbivores were negatively correlated with increased Cd concentrations in plant tissues. In addition, the feeding preferences of the two leaf herbivore species were strongly repelled by leaf discs from Cd-treated plants with Spd supplement. The results suggested that the application of exogenous Spd at a certain dose could enhance elemental defense of plants against herbivory.

Effects of bistrifluron resistance on the biological traits of Spodoptera litura (Fab.) (Noctuidae: Lepidoptera).

Spodoptera litura is one of the major insect pests of vegetables in Sichuan, China, and it has developed serious resistance to many traditional chemical insecticides. In this present study, S. litura individuals collected from five regions in Sichuan in 2017, which were detected the resistance to bistrifluron. We found that the resistance ratios (RRs) bistrifluron were in the ranges of 15.9- to 77.7-fold, respectively, compared with a laboratory susceptible (Lab-HN) strain. To elucidate the effect of bistrifluron on the life history parameters of S. litura, we established the bistrifluron-susceptible (Bis-UNSEL) and bistrifluron-resistant (Bis-SEL) strains based on the DY field population with the highest RR of 77.7-fold among the all field populations to bistrifluron compared with a laboratory Lab-HN strain. The results show that the Bis-SEL strain developed a 113.8-fold RR while that of the Bis-UNSEL strain was almost equal to that of the Lab-HN strain (with overlapping confidence intervals (CI) of their LC50 values, P < 0.05). Meanwhile, the life span of the egg and pupa stage of the Bis-UNSEL strain was prolonged, and the strain also showed shorter adult duration and lower fecundity. Demographic characteristics, such as the intrinsic rate of increase (r), finite rate of increase (λ) and net reproductive rate (R0) in the Bis-SEL strain were significantly higher than the Bis-UNSEL strain, whereas the mean generation time (T) and gross reproduction rate (GRR) were lower. The results clearly showed that the developing resistance of bistriflueon on S. litura promoted its population growth. Therefore, the present study could provide useful informations for determining potential management methods to prevent the development of bistrifluron resistance in S. litura.

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.

Monitoring and biochemical characterization of beta-cypermethrin resistance in Spodoptera exigua (Lepidoptera: Noctuidae) in Sichuan Province, China.

The beet armyworm Spodoptera exigua, a major pest affecting numerous cultivated crops in China, has developed a serious resistance to many traditional chemical insecticides. The resistance levels of the field-collected populations from different districts in Sichuan Province, China, to nine insecticides were detected with a diet-incorporation bioassay. Compared to the Lab-ZN strain, five (in 2014) and three (in 2016) field populations displayed either high or extremely high levels of resistance to beta-cypermethrin. All the field populations collected in 2014 were susceptible to emamectin benzoate, hexaflumuron, methoxyfenozide, chlorantraniliprole, cyantraniliprole and indoxacarb but exhibited low or moderate levels of resistance to abamectin. The resistances of field populations collected in 2016 were significantly higher than two years earlier, especial for chlorantraniliprole and cyantraniliprole with RRs rising from 173.4- to 582.6-fold and 175.3- to 287.6-fold, respectively, even though the field populations had retained moderate or low levels of resistance to chlorpyrifos and hexaflumuron. The synergism experiment revealed that the resistance of the LS16 population to beta-cypermethrin may be mainly related to cytochrome P450 monooxygenases (P450s), which was responsible for the highest increase ratio of 37.97-fold, for piperonyl butoxide, rather than either carboxylesterase (CarE) or glutathione S-transferase (GST). The cytochrome P450 ethoxycoumarin O-deethylase activity of the LS16 population was also the strongest among the treatments (P < 0.05). Non-denaturing polyacrylamide gel electrophoresis (native PAGE) indicated that enhanced E11, E13 and E15-E16 bands in the LS16 population likely contribute to the development of resistance to beta-cypermethrin.

The Effect of Chemical Composition and Bioactivity of Several Essential Oils on Tenebrio molitor (Coleoptera: Tenebrionidae).

The major chemical components of four essential oils (EOs) extracted from dry leaves of Citrus limonum, Cymbopogon citratus, Litsea cubeba, and Muristica fragrans were analyzed with gas chromatograph-mass spectrometer and their fumigant, contact, and repellent activities against 10th instar and adults of Tenebrio molitor were also assayed. The results indicated that the major constituents of C. limonum and Cy. citrates were D-limonene (38.22%) and 3,7-dimethyl-6-octenal (26.21%), while which of L. cubeba and M. fragrans were (E)-3, 7-dimethyl-2, 6-octadienal (49.78%) and (E)-cinnamaldehyde (79.31%), respectively. Contact activities of L. cubeba and C. limonum with LC50 values of 21.2 and 13.9 µg/cm(2) at 48 h and repellence activities (>89.0% repellence indexes) (P < 0.05) at 12 h on 10th instar were better than those of the other two EOs. Nevertheless, the fumigation activities of L. cubeba on 10th instar and adults (LC50 = 2.7, 3.7 µl/liter) were stronger than those of C. limonum (LC50 = 10.9, 12.0 µl/liter) at 96 h and significant (not overlapping confidence intervals). The EOs of L. cubeba and C. limonum have clearly elongated the growth and development of larvae, egg, and slightly shorten pupae and adults of T. molitor compared with the control. The mainly active ingredients of L. cubeba and C. limonum, including D-limonene and ß-pinene, were demonstrated to coinhibit the actives of AChE and enhance the toxicities on 10th instar of T. molitor. These results indicate that the EOs of L. cubeba and C. limonum could have great potential as botanical insecticides against T. molitor.

Fumigant, contact, and repellent activities of essential oils against the darkling beetle, Alphitobius diaperinus.

The fumigant, contact, and repellent activities of four essential oils extracted from Citrus limonum (Sapindales: Rutaceae), Litsea cubeba (Laurales: Lauraceae), Cinnamomum cassia, and Allium sativum L. (Asparagales: Alliaceae) against 6th instars and adults of the darkling beetle, Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae), one of the main pests of materials and products of Juncus effuses L. (Poales: Juncaceae) during the storage period, were assayed, and chemical ingredients were analyzed with gas chromatography-mass spectrometry in this study. While the major ingredients found in C. limonum and C. cassia were limonene and (E)-cinnamaldehyde, the main constituents of L. cubea were D-limonene, (E)-3,7-dimethyl-,2,6-octadienal, (Z)-3,7-dimethyl,2 ,6-octadienal, and diallyl disulphide (18.20%), while the main constituents of and A. sativum were di-2-propenyl trisulfide and di-2-propenyl tetrasulfide. The fumigation activities of A. sativum and C. limonum on A. diaperinus adults were better than those of the other two essential oilss. The toxicities of A. sativum and C. limonum were almost equitoxic at 96 hr after treatment. Essential oils from Allium sativum and L. cubeba also showed good contact activities from 24 hr to 48 hr, and toxicities were almost equitoxic 48 hr posttreatment. The repellent activities of A. sativum and L. cubeba oils on 6th instars were also observed, showing repellence indexes of 90.4% and 88.9% at 12 hr after treatment, respectively. The effects of A. sativum on AChE activity of 6th instars of A. diaperinus were strongest compared to the other essential oils, followed by C. limonum, L. cubeba, and C. cassia. These results suggest that the essential oils of C. limonum and A. sativum could serve as effective control agents of A. diaperinus.

Discovery of Novel Potential Insecticide-Resistance Mutations in Spodoptera frugiperda.

The fall armyworm (FAW), Spodoptera frugiperda, is a worldwide agricultural pest that invaded China in 2018, and has developed resistance to multiple insecticides. The evolution of insecticide resistance is facilitated by mutations of target genes responsible for conferring resistance. In this study, amplicon sequencing analyzed 21 sites in six resistance genes. In addition to known mutations, unknown variants were also found, including novel variants: F290C (ace-1 gene, 0.1% frequency), I1040T/V (CHSA gene, 0.1% frequency), A309T (GluCl gene, 0.1% frequency), and I4790T/V (RyR gene, 0.1% frequency). Additionally, molecular docking was employed to investigate the impact of the aforementioned new mutations on insecticide binding to proteins. The analyses indicated that the binding abilities were reduced, similar to the resistance mutations that were reported, implying these novel mutations may confer transitional resistance. This study may provide a foundation for understanding the functions of these novel mutations in the evolutionary processes that drive the emergence of insecticide resistance in this invasive species.

Corrigendum: Effects of chlorantraniliprole on the life history traits of fall armyworm Spodoptera frugiperda (Lepidoptera: Noctuidae).

[This corrects the article DOI: 10.3389/fphys.2023.1155455.].