Key role of the ryanodine receptor I4790K mutation in mediating diamide resistance in Plutella xylostella.

The diamondback moth Plutella xylostella, a global insect pest of cruciferous vegetables, has evolved resistance to many classes of insecticides including diamides. Three point mutations (I4790M, I4790K, and G4946E) in the ryanodine receptor of P. xylostella (PxRyR) have been identified to associate with varying levels of resistance. In this study, we generated a knockin strain (I4790K-KI) of P. xylostella, using CRISPR/Cas9 to introduce the I4790K mutation into PxRyR of the susceptible IPP-S strain. Compared to IPP-S, the edited I4790K-KI strain exhibited high levels of resistance to both anthranilic diamides (chlorantraniliprole 1857-fold, cyantraniliprole 1433-fold) and the phthalic acid diamide flubendiamide (>2272-fold). Resistance to chlorantraniliprole in the I4790K-KI strain was inherited in an autosomal and recessive mode, and genetically linked with the I4790K knockin mutation. Computational modeling suggests the I4790K mutation reduces the binding of diamides to PxRyR by disrupting key hydrogen bonding interactions within the binding cavity. The approximate frequencies of the 4790M, 4790K, and 4946E alleles were assessed in ten geographical field populations of P. xylostella collected in China in 2021. The levels of chlorantraniliprole resistance (2.3- to 1444-fold) in these populations were significantly correlated with the frequencies (0.017-0.917) of the 4790K allele, but not with either 4790M (0-0.183) or 4946E (0.017-0.450) alleles. This demonstrates that the PxRyR I4790K mutation is currently the major contributing factor to chlorantraniliprole resistance in P. xylostella field populations within China. Our findings provide in vivo functional evidence for the causality of the I4790K mutation in PxRyR with high levels of diamide resistance in P. xylostella, and suggest that tracking the frequency of the I4790K allele is crucial for optimizing the monitoring and management of diamide resistance in this crop pest.

Helicoverpa armigera ATP-binding cassette transporter ABCA2 is a functional receptor of Bacillus thuringiensis Cry2Ab toxin.

Crystalline (Cry) proteins from the bacterium Bacillus thuringiensis (Bt) are widely used in transgenic crops to control important insect pests. Bt crops have many benefits compared with traditional broad-spectrum insecticides, including improved pest control with reduced negative impacts on off-target organisms and fewer environmental consequences. Transgenic corn and cotton producing Cry2Ab Bt toxin are used globally to control several major lepidopteran pests, including the cotton bollworm, Helicoverpa armigera. Resistance to the Cry2Ab toxin and to Bt crops producing Cry2Ab is associated with mutations in the midgut ATP-binding cassette transporter ABCA2 gene in several lepidopterans. Gene-editing knockout has further shown that ABCA2 plays an important functional role in Cry2Ab intoxication. However, the precise role of ABCA2 in the mode of action of Cry2Ab has yet to be reported. Here, we used two in vitro expression systems to study the roles of the H. armigera ABCA2 (HaABCA2) protein in Cry2Ab intoxication. Cry2Ab bound to cultured Sf9 insect cells producing HaABCA2, resulting in specific and dose-dependent susceptibility to Cry2Ab. In contrast, Sf9 cells expressing recombinant mutant proteins missing at least one of the extracellular loop regions 1, 3, 4, and 6 or the intracellular loop containing nucleotide-binding domain 1 lost susceptibility to Cry2Ab, indicating these regions are important for receptor function. Consistent with these results, Xenopus laevis oocytes expressing recombinant HaABCA2 showed strong ion membrane flux in the presence of Cry2Ab, suggesting that HaABCA2 is involved in promoting pore formation during Cry2Ab intoxication. Together with previously published data, our results support HaABCA2 being an important receptor of Cry2Ab where it functions to promote intoxication in H. armigera.

Varying contributions of three ryanodine receptor point mutations to diamide insecticide resistance in Plutella xylostella.

BACKGROUND: Although decoding the molecular mechanisms underlying insecticide resistance has often proven difficult, recent progress has revealed that specific mutations in the ryanodine receptor (RyR) of the diamondback moth, Plutella xylostella, can confer resistance to diamide insecticides. The extent to which specific RyR mutations contribute to the diamide resistance phenotype, the associated genetic traits and fitness costs remain limited. RESULTS: Three field-evolved PxRyR mutations (G4946E, I4790 M, and I4790 K) were respectively introgressed into a common susceptible background strain (IPP-S) of P. xylostella with marker-assisted backcrossing. The mutations alone can result in moderate to high levels of resistance to five commercial diamides (flubendiamide, chlorantraniliprole, cyantraniliprole, tetraniliprole, and cyclaniliprole), and the resistance intensity mediated by the three mutations was hierarchical in order of I4790 K (1199- to >2778-fold) > G4946E (39- to 739-fold) > I4790 M (16- to 57-fold). Flubendiamide resistance was autosomal and incompletely recessive, and was significantly linked with the introgressed mutations in the three constructed strains. In addition, the resistance levels to flubendiamide of hybrid progeny from any two resistant strains fell in between the status of their parents. Furthermore, by comparing the net replacement rate, the fitness of 4946E, 4790 M and 4790 K strains were 0.77, 0.93 and 0.92 relative to the IPP-S strain, respectively. CONCLUSION: Three independent PxRyR mutations confer varying degrees of resistance to diamides in P. xylostella. Among the three mutations, I4790 K confers highest levels of resistance (> 1000-fold) to all five commercial diamides. The findings can guide resistance management practices for diamides in P. xylostella and other arthropods.

Independent and Synergistic Effects of Knocking out Two ABC Transporter Genes on Resistance to Bacillus thuringiensis Toxins Cry1Ac and Cry1Fa in Diamondback Moth.

Insecticidal proteins from Bacillus thuringiensis (Bt) are used widely in sprays and transgenic crops to control insect pests. However, evolution of resistance by pests can reduce the efficacy of Bt toxins. Here we analyzed resistance to Bt toxins Cry1Ac and Cry1Fa in the diamondback moth (Plutella xylostella), one of the world's most destructive pests of vegetable crops. We used CRISPR/Cas9 gene editing to create strains with knockouts of the ATP-binding cassette (ABC) transporter genes PxABCC2, PxABCC3, or both. Bioassay results show that knocking out either gene alone caused at most 2.9-fold resistance but knocking out both caused >10,320-fold resistance to Cry1Ac and 380-fold resistance to Cry1Fa. Cry1Ac resistance in the double knockout strain was recessive and genetically linked with the PxABCC2/PxABCC3 loci. The results provide insight into the mechanism of cross-resistance to Cry1Fa in diamondback moth. They also confirm previous work with this pest showing that mutations disrupting both genes cause higher resistance to Cry1Ac than mutations affecting either PxABCC2 or PxABCC3 alone. Together with previous work, the results here highlight the value of using single and multiple gene knockouts to better understand the independent and synergistic effects of putative Bt toxin receptors on resistance to Bt toxins.

Disruption of nicotinic acetylcholine receptor α6 mediated by CRISPR/Cas9 confers resistance to spinosyns in Plutella xylostella.

BACKGROUND: The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system provides some advantages over other reverse genetic techniques to investigate the causal relationship between insecticide resistance phenotype and candidate gene. Several studies published to date point to the nicotinic acetylcholine receptor (nAChR) α6 subunit strongly associated with spinosyns resistance in insects, including Plutella xylostella. However, reverse genetic verification of the P. xylostella nAChRα6 has not yet been achieved via an in vivo approach. RESULTS: Here, we successfully constructed a homozygous strain (Pxα6-KO) with a 2-nt deletion mutation of nAChRα6 by CRISPR/Cas9 coupled with non-homologous end joining approach in P. xylostella. The manipulated mutation results in a frame shift in the open reading frame of transcripts, which produces a predicted protein truncated in the TM3-TM4 loop region. When compared to the background strain IPP-S, the knockout strain Pxα6-KO exhibited 229- and 1462-fold resistance to spinosad and spinetoram, respectively, but no or limited (resistance ratios <3-fold) effects on the toxicities of imidacloprid, abamectin, ß-cypermethrin, indoxacarb, metaflumizone and chlorantraniliprole. Furthermore, the mode of inheritance of the acquired spinetoram resistance was autosomal recessive and significantly linked with the 2-nt deletion mutation of nAChRα6 in the Pxα6-KO strain. CONCLUSION: In vivo functional investigation demonstrates the causality of the Pxα6 truncating mutation with high levels of resistance to spinosyns in P. xylostella. Our results suggest the Pxα6-KO strain underlies an autosomal, recessive mode of inheritance for spinetoram resistance, and reinforces the association of this gene to the mode of action of spinosyns. © 2019 Society of Chemical Industry.

Long-term monitoring and characterization of resistance to chlorfenapyr in Plutella xylostella (Lepidoptera: Plutellidae) from China.

BACKGROUND: Plutella xylostella (L.) is a worldwide pest of crucifer crops. Chlorfenapyr has been used extensively to manage several insect pests in vegetables, including P. xylostella. In this study, 48 field populations were collected from 11 geographic locations of China from 2008 to 2017 for resistance monitoring to chlorfenapyr. Inheritance and synergism of chlorfenapyr resistance were characterized in a field-derived population. RESULTS: Compared with the susceptible SZ strain, 33 of 39 populations collected from Eastern China were susceptible or developed low levels of resistance to chlorfenapyr (< 10-fold), five populations developed mid-level resistance (11-33-fold), and only one population had high level resistance (208-fold). However, seven of nine populations sampled from Southern China evolved 136-334-fold resistance. Interestingly, the dynamic of the median lethal concentration (LC50 ) values from four continuous sampling sites (NJ, HF, JN and KS) showed a sharp increase in 2009, which implies that adults may have immigrated from chlorfenapyr-resistant regions. A highly resistant field population (GZ15) was further studied. By crossing the GZ15 and susceptible SZ strains we showed that resistance to chlorfenapyr was autosomally inherited and incompletely dominant. Synergism bioassays revealed that metabolic detoxification might not be responsible for chlorfenapyr resistance in the GZ15 population. CONCLUSIONS: Some field populations of P. xylostella from China have evolved high levels of resistance to chlorfenapyr. Field-evolved resistance to chlorfenapyr is autosomal and dominant in the GZ15 population. These findings provide useful information for the design of effective resistance management strategies against P. xylostella in the field. © 2018 Society of Chemical Industry.

[Effect of MAPK/NF-kappaB signaling pathway on extracellular release of HMGB1 induced by hypoxia in laryngeal Hep-2 carcinoma cells].

OBJECTIVE: To investigate the extracellular release of high mobility group box 1 (HMGB1) in laryngeal Hep-2 carcinoma cells induced by hypoxia and its possible mechanism. METHOD: The changes of HMGB1 concentration in the culture medium as well as HMGB1 protein and mRNA expression in Hep-2 cells were investigated after the cells were cultured with 1% O2 for different durations. Inhibitory effects of MAPK pathway inhibitors (PD98059. SP600125, and SB202190) and nuclear NF-kappaB pathway inhibitor (PDTC) with various concentrations on extracellular HMGB1 release were observed in hypoxia-induced Hep-2 cells. The HMGB1 concentration and HMGB1 protein expression were measured by enzyme-linked immunosorbent assay (ELISA) and western blot, respectively. The HMGB1 mRNA expression was determined by real-time quantitative PCR(RT-PCR). RESULT: The HMGB1 concentration in the culture medium and the HMGB1 protein expression in Hep-2 cells increased after the cells were subjected to hypoxia culture for 12 h in a time-dependent manner. The level of HMGB1 mRNA expression in Hep-2 cells increased after the cells were induced by hypoxia for 6h PD98059 and SP600125 with 20 micromol/ L and PDTC with 50 mg/L partly inhibited extracellular release of HMGB1 in hypoxia-cultured Hcp-2 cells. CONCLUSION: Hypoxia induces laryngeal carcinoma cells to release HMGH1. which may be related to MAPK/NF-kappaB signaling pathway.