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.

A General Signal Pathway to Regulate Multiple Detoxification Genes Drives the Evolution of Helicoverpa armigera Adaptation to Xenobiotics.

The study of insect adaptation to the defensive metabolites of host plants and various kinds of insecticides in order to acquire resistance is a hot topic in the pest-control field, but the mechanism is still unclear. In our study, we found that a general signal pathway exists in H. armigera which can regulate multiple P450s, GSTs and UGTs genes to help insects decrease their susceptibility to xenobiotics. Knockdown of HaNrf2 and HaAhR expression could significantly increase the toxicity of xenobiotics to H. armigera, and simultaneously decrease the gene expression of P450s, GSTs and UGTs which are related to the xenobiotic metabolism and synthesis of insect hormone pathways. Then, we used EMSA and dual luciferase assay to verify that a crosstalk exists between AhR and Nrf2 to regulate multiple P450s, GSTs and UGTs genes to mediate H. armigera susceptibility to plant allelochemicals and insecticides. The detoxification genes' expression network which can be regulated by Nrf2 and AhR is still unknown, and there were also no reports about the crosstalk between AhR and Nrf2 that exist in insects and can regulate multiple detoxification genes' expression. Our results provide a new general signaling pathway to reveal the adaptive mechanism of insects to xenobiotics and provides further insight into designing effective pest-management strategies to avoid the overuse of insecticides.

Corynebacterium sp. 2-TD Mediated Toxicity of 2-Tridecanone to Helicoverpa armigera.

Cotton bollworm (Helicoverpa armigera) is a Lepidopteran noctuid pest with a global distribution. It has a wide range of host plants and can harm cotton, tomato, tobacco, and corn, as well as other crops. H. armigera larvae damage the flower buds, flowers, and fruits of tomato and cause serious losses to tomato production. Tomato uses the allelochemical 2-tridecanone to defend against this damage. So far, there have been no reports on whether the adaptation of H. armigera to 2-tridecanone is related to its symbiotic microorganisms. Our study found that Corynebacterium sp. 2-TD, symbiotic bacteria in H. armigera, mediates the toxicity of the 2-tridecanone to H. armigera. Corynebacterium sp. 2-TD, which was identified by 16S rDNA gene sequence analysis, was screened out using a basal salt medium containing a unique carbon source of 2-tridecanone. Then, Corynebacterium sp. 2-TD was confirmed to be distributed in the gut of H. armigera by quantitative PCR (qPCR) and fluorescence in situ hybridization (FISH). The survival rate of H. armigera increased by 38.3% under 2-tridecanone stress after inoculation with Corynebacterium sp. 2-TD. The degradation effect of Corynebacterium sp. 2-TD on 2-tridecanone was verified by ultra-high-performance liquid chromatography (UPLC). Our study is the first to report the isolation of gut bacteria that degrade 2-tridecanone from the important agricultural pest H. armigera and to confirm bacterial involvement in host adaptation to 2-tridecanone, which provides new insights into the adaptive mechanism of agricultural pests to host plants.

Detection of Chitin Synthase Mutations in Lufenuron-Resistant Spodoptera frugiperda in China.

Spodoptera frugiperda (J. E. Smith), is commonly known as fall armyworm, native to tropical and subtropical regions of America, is an important migratory agricultural pest. It is important to understand the resistance and internal mechanism of action of S. frugiperda against lufenuron in China. Lufenuron is one of the main insecticides recommended for field use in China and has a broad prospect in the future. We conducted a bioassay using the diet-overlay method and found that the current S. frugiperda in China are still at a low level of resistance to lufenuron. Secondly, we examined whether the mutation I1040M (I1042M in Plutella xylostella), associated with lufenuron resistance, was produced in the field. And then we tested the expression of chitin synthase SfCHSA and SfCHSB in different tissues, and the changes of these two genes after lufenuron induction. The results showed that there is still no mutation generation in China and there is a significant change in the expression of SfCHSA under the effect of lufenuron. In conclusion, our study suggests that field S. frugiperda populations in 2019 and 2020 were less resistant to lufenuron. In fall armyworm, chitin synthases included SfCHSA and SfCHSB genes, and after induction treatment with lufenuron, the expression of the SfCHSA gene was significantly increased. In SfCHSA, no mutation has been detected in the site associated with lufenuron resistance. Secondly, in S. frugiperda larvae, the SfCHSA gene was the highest in the head of the larvae, followed by the integument; while the SfCHSB gene was mainly concentrated in the midgut. Therefore, we believe that the SfCHSA gene plays a greater role in the resistance of S. frugiperda to lufenuron than the SfCHSB gene. It is worth noting that understanding the level of resistance to lufenuron in China, the main mechanism of action of lufenuron on larvae, and the mechanism of resistance to lufenuron in S. frugiperda will help in crop protection as well as in extending the life span of this insecticide.

Detection of ryanodine receptor target-site mutations in diamide insecticide-resistant Spodoptera frugiperda in China.

Spodoptera frugiperda (Lepidoptera: Noctuidae) is a widely distributed pest of corn. Since it invaded China in 2018, it has caused serious damage to local corn production. Chlorantraniliprole, an anthranilic diamide insecticide, has been widely used to control lepidopteran pests. Tetrachloropyramid is a new allosteric modulator insecticide developed based on chlorantraniliprole, so it has a similar mechanism and insecticidal effect. In this study, we investigated resistance levels to chlorantraniliprole and tetrachloropyramid in S. frugiperda from 13 populations in China. Among the populations tested, the relative highest resistance to chlorantraniliprole occurred in the Guangzhou population, and the most susceptible to chlorantraniliprole was found in the Wuhan population. The lethal dosage LD50 value of the Guangzhou population against chlorantraniliprole was 27.8-fold higher than that of the Wuhan population. Minimal differences were observed among S. frugiperda populations in terms of sensitivity to tetrachloropyramid. Heterozygous mutations at the I4734 site of the ryanodine receptor (RyR) were found, while no mutations were found in the G4891 site. The mutations were detected in only two of the 786 individuals analyzed, one from the Qinzhou population and other from the Anshun population (frequency below 2% in both cases). There were no significant differences in the expression levels of RyR between Guangzhou and Wuhan populations. In summary, our results indicate that: (i) S. frugiperda has low resistance levels to diamide insecticides in China; and (ii) the differences in relative resistance among the 13 populations analyzed are not caused by the mutations in RyR or the expression of RyR.

Cellular Redox-Related Transcription Factor Nrf2 Mediation of HaTrf Response to Host Plant Allelochemical 2-Tridecanone in Helicoverpa armigera.

Despite there being a number of excellent studies on detoxification enzyme-mediated interaction between insect and plant allelochemical, there are no reports on the pathway of the transferrin effect in insect response to host plant allelochemical. Our research indicates that Helicoverpa armigera transferrin (HaTrf) inhibited the apoptotic cell death treated by 2-tridecanone, a host plant allelochemical present in tomato species (Lycopersicon hirsutum f. glabratum), by cellular redox-related transcription factor nuclear factor erythroid-2-related factor 2 (Nrf2). Nrf2 can defend organisms against the detrimental effects of oxidative stress and play pivotal roles in preventing host plant allelochemical-related toxicity. This study explains how HaTrf inhibited the apoptotic cell death during exposure to host plant allelochemical 2-tridecanone and provides a novel view on transferrin and its anti-apoptotic role in plant-insect interactions.