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The interactions between insects and their bacterial symbionts are shaped by a variety of abiotic factors, including temperature. As global temperatures continue to break high records, a great deal of uncertainty surrounds how agriculturally important insect pests and their symbionts may be affected by elevated temperatures, and its implications for future pest management. In this study, we examine the role of bacterial symbionts in the brown planthopper Nilaparvata lugens response to insecticide (imidacloprid) under different temperature scenarios. Our results reveal that the bacterial symbionts orchestrate host detoxification metabolism via the CncC pathway to promote host insecticide resistance, whereby the symbiont-inducible CncC pathway acts as a signaling conduit between exogenous abiotic stimuli and host metabolism. However, this insect-bacterial partnership function is vulnerable to high temperature, which causes a significant decline in host-bacterial content. In particular, we have identified the temperature-sensitive Wolbachia as a candidate player in N. lugens detoxification metabolism. Wolbachia-dependent insecticide resistance was confirmed through a series of insecticide assays and experiments comparing Wolbachia-free and Wolbachia-infected N. lugens and also Drosophila melanogaster. Together, our research reveals elevated temperatures negatively impact insect-bacterial symbiosis, triggering adverse consequences on host response to insecticide (imidacloprid) and potentially other xenobiotics.

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.

Sogatella furcifera (Horvath) is a notorious pest in most rice-producing regions of Asia, and evidence of resistance of S. furcifera to various insecticides has been documented in many areas. Given that the risk of insecticide resistance has spread, prospective monitoring should be expedited. Diagnostic tools for the rapid and accurate assessment of insecticide resistance are urgently needed to implement pest control and effective insecticide resistance management strategies. In the present study, a diagnostic kit based on the glass vial bioassay for the rapid detection of resistance to imidacloprid, nitenpyram, clothianidin, dinotefuran, thiamethoxam, isoprocarb and chlorpyrifos in S. furcifera was developed. The detection results can be obtained in 1 h, and the determination of ≥ 90% mortality indicates sensitivity to insecticides. The insecticide resistance levels diagnosed by the kit were consistent with the results of the rice seedling dip method in field populations of S. furcifera. Moreover, the accuracy of the diagnosis of nitenpyram and chlorpyrifos resistance was further verified by field trials. The insecticide resistance diagnostic kit for S. furcifera, as a user-friendly tool (portable, short detection time), can be easily popularized. Its excellent performance qualifies the kit as a reliable screening tool to determine effective insecticides and a convenient addition to integrated pest management programs.

Carboxylesterases (CarEs) represent one of the major detoxification enzyme families involved in insecticide resistance. However, the function of specific CarE genes in insecticide resistance is still unclear in the insect Nilaparvata lugens (Stål), a notorious rice crop pest in Asia. In this study, a total of 29 putative CarE genes in N. lugens were identified, and they were divided into seven clades; further, the β-esterase clade was significantly expanded. Tissue-specific expression analysis found that 17 CarE genes were abundantly distributed in the midgut and fat body, while 12 CarE genes were highly expressed in the head. The expression of most CarE genes was significantly induced in response to the challenge of nitenpyram, triflumezopyrim, chlorpyrifos, isoprocarb and etofenprox. Among these, the expression levels of NlCarE2, NlCarE4, NlCarE9, NlCarE17 and NlCarE24 were increased by each insecticide. Real-time quantitative polymerase chain reaction and RNA interference assays revealed the NlCarE1 gene to be a candidate gene mainly involved in nitenpyram resistance, while simultaneously silencing NlCarE1 and NlCarE19 produced a stronger effect than silencing either one individually, suggesting a cooperative relationship in resistance formation. These findings lay the foundation for further clarification of insecticide resistance mediated by CarE in N. lugens.

The accumulation of fluoride in tea leaves from various cultivars exhibits significant differences. However, the molecular basis and mechanism remain largely unknown. Here, we reported that two genes of CsFEX (fluoride export genes in Camellia sinensis), CsFEX1 and CsFEX2, transport fluoride out of cells, alleviate the cellular fluoride toxin, and rescue the yeast mutant (FEX1ΔFEX2Δ) and Arabidopsis mutant (fex), as their efflux activities are coupled with proton gradients. Further analysis found that CsFEX1 and CsFEX2 localize to the plasma membrane both in yeast and Arabidopsis cells. CsFEX2 is more effective to reduce fluoride toxicity in yeast and Arabidopsis compared with CsFEX1 even at low pH. CsFEX2 induced by fluoride treatment is around tenfold higher in a low-fluoride cultivar (Yunkang 10) than that in a high-fluoride cultivar (Pingyang Tezaocha), suggesting that CsFEX2 possibly plays a critical role in reducing fluoride accumulation in tea leaves.

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.

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.

In this study, the sensitivity of 20 field populations of Chilo suppressalis (Walker) from five provinces in China to seven insecticides was evaluated during 2016–2018. The results indicated that 20 field populations of C. suppressalis had evolved moderate to high levels of resistance to triazophos (RR 64.5–461.3) and chlorpyrifos (RR 10.1–125.0). Furthermore, C. suppressalis exhibited low to moderate levels of resistance to abamectin (RR 6.5–76.5) and decreased susceptibility to cyantraniliprole (RR 1.0–34.0). The population collected from Nanchang in Jiangxi Province (JXNC) showed high resistance to chlorantraniliprole (RR 148.3–294.3), and other geographical populations remained susceptible to moderate levels of resistance (RR 1.0–37.5). In contrast, C. suppressalis remained susceptible to low levels of resistance to spinetoram (RR 1.0–6.7) and spinosad (RR 1.0–4.6). Significant correlations were found between the Log LC50 values of chlorantraniliprole and cyantraniliprole, chlorpyrifos and triazophos, as well as cyantraniliprole and chlorpyrifos and triazophos. Similarly, significant correlations were found among abamectin, chlorpyrifos, and triazophos. In addition, a significant correlation was also observed between the activity of the detoxification enzymes and the log LC50 values of chlorantraniliprole, cyantraniliprole, abamectin, chlorpyrifos, and triazophos. The findings provide an important reference for implementing effective resistance management strategies and the development of new insecticides in insect pest control.

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.

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 N. lugens' resistance 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 downregulation 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. This article is protected by copyright. All rights reserved.

A leaf-to-caryopsis abscisic acid transport mechanism ensures normal seed development in response to variable temperatures., Long-distance transport of the phytohormone abscisic acid (ABA) has been studied for ~50 years, yet its mechanistic basis and biological significance remain very poorly understood. Here, we show that leaf-derived ABA controls rice seed development in a temperature-dependent manner and is regulated by defective grain-filling 1 (DG1), a multidrug and toxic compound extrusion transporter that effluxes ABA at nodes and rachilla. Specifically, ABA is biosynthesized in both WT and dg1 leaves, but only WT caryopses accumulate leaf-derived ABA. Our demonstration that leaf-derived ABA activates starch synthesis genes explains the incompletely filled and floury seed phenotypes in dg1. Both the DG1-mediated long-distance ABA transport efficiency and grain-filling phenotypes are temperature sensitive. Moreover, we extended these mechanistic insights to other cereals by observing similar grain-filling defects in a maize DG1 ortholog mutant. Our study demonstrates that rice uses a leaf-to-caryopsis ABA transport–based mechanism to ensure normal seed development in response to variable temperatures.

Nitrate-induced Ca2+ signaling is crucial for the primary nitrate response in plants. However, the molecular mechanism underlying the generation of the nitrate-specific calcium signature remains unknown. We report here that a cyclic nucleotide-gated channel (CNGC) protein, CNGC15, and the nitrate transceptor (NRT1.1) constitute a molecular switch that controls calcium influx depending on nitrate levels. The expression of CNGC15 is induced by nitrate, and its protein is localized at the plasma membrane after establishment of young seedlings. We found that disruption of CNGC15 results in the loss of the nitrate-induced Ca2+ signature (primary nitrate response) and retards root growth, reminiscent of the phenotype observed in the nrt1.1 mutant. We further showed that CNGC15 is an active Ca2+-permeable channel that physically interacts with the NRT1.1 protein in the plasma membrane. Importantly, we discovered that CNGC15–NRT1.1 interaction silences the channel activity of the heterocomplex, which dissociates upon a rise in nitrate levels, leading to reactivation of the CNGC15 channel. The dynamic interactions between CNGC15 and NRT1.1 therefore control the channel activity and Ca2+ influx in a nitrate-dependent manner. Our study reveals a new nutrient-sensing mechanism that utilizes a nutrient transceptor–channel complex assembly to couple nutrient status to a specific Ca2+ signature.

Unloading sugar from sink phloem by transporters is complex and much remains to be understood about this phenomenon in the watermelon fruit. Here, we report a novel vacuolar sugar transporter (ClVST1) identified through map-based cloning and association study, whose expression in fruit phloem is associated with accumulation of sucrose (Suc) in watermelon fruit. ClVST197 knockout lines show decreased sugar content and total biomass, whereas overexpression of ClVST197 increases Suc content. Population genomic and subcellular localization analyses strongly suggest a single-base change at the coding region of ClVST197 as a major molecular event during watermelon domestication, which results in the truncation of 45 amino acids and shifts the localization of ClVST197 to plasma membranes in sweet watermelons. Molecular, biochemical and phenotypic analyses indicate that ClVST197 is a novel sugar transporter for Suc and glucose efflux and unloading. Functional characterization of ClVST1 provides a novel strategy to increase sugar sink potency during watermelon domestication.

Root nodule symbiosis enables nitrogen fixation in legumes and, therefore, improves crop production for sustainable agriculture1,2. Environmental nitrate levels affect nodulation and nitrogen fixation, but the mechanisms by which legume plants modulate nitrate uptake to regulate nodule symbiosis remain unclear1. Here, we identify a member of the Medicago truncatula nitrate peptide family (NPF), NPF7.6, which is expressed specifically in the nodule vasculature. NPF7.6 localizes to the plasma membrane of nodule transfer cells (NTCs), where it functions as a high-affinity nitrate transporter. Transfer cells show characteristic wall ingrowths that enhance the capacity for membrane transport at the apoplasmic-symplasmic interface between the vasculature and surrounding tissues3. Importantly, knockout of NPF7.6 using CRISPR-Cas9 resulted in developmental defects of the nodule vasculature, with excessive expansion of NTC plasma membranes. npf7.6 nodules showed severely compromised nitrate responsiveness caused by an attenuated ability to transport nitrate. Moreover, npf7.6 nodules exhibited disturbed nitric oxide homeostasis and a notable decrease in nitrogenase activity. Our findings indicate that NPF7.6 has been co-opted into a regulatory role in nodulation, functioning in nitrate uptake through NTCs to fine-tune nodule symbiosis in response to fluctuating environmental nitrate status. These observations will inform efforts to optimize nitrogen fixation in legume crops.

Increasing carbon dioxide (CO₂) levels in the atmosphere have caused global metabolic changes in diverse plant species. CO₂ is not only a carbon donor for photosynthesis but also an environmental signal that regulates stomatal movements and thereby controls plant-water relationships and carbon metabolism. However, the mechanism underlying CO₂ sensing in stomatal guard cells remains unclear. Here we report characterization of Arabidopsis RESISTANT TO HIGH CO₂ (RHC1), a MATE-type transporter that links elevated CO₂ concentration to repression of HT1, a protein kinase that negatively regulates CO₂-induced stomatal closing. We also show that HT1 phosphorylates and inactivates OST1, a kinase which is essential for the activation of the SLAC1 anion channel and stomatal closing. Combining genetic, biochemical and electrophysiological evidence, we reconstituted the molecular relay from CO₂ to SLAC1 activation, thus establishing a core pathway for CO₂ signalling in plant guard cells.