Insect herbivores rely on glutathione S-transferases (GST) to cope with host-derived secondary metabolites, yet the specific role of individual GST isoforms in mediating host adaptation remains poorly understood. Here, we characterized the sigma-class GST gene NlGSTs3 and its role in host plant adaptation of the brown planthopper Nilaparvata lugens. NlGSTs3 was predominantly expressed in the fat body and testes. RNAi-mediated knockdown of NlGSTs3 significantly reduced survival and feeding performance on rice plants and specifically increased susceptibility to ferulic acid and resorcinol. Molecular docking predicted favorable interactions between NlGSTs3 and these two compounds, which was further supported by in vitro catalytic assays showing recombinant NlGSTs3 mediates the depletion of ferulic acid and resorcinol. This work demonstrates that NlGSTs3 contributes to the adaptation of N. lugens to rice chemical defenses through the substrate-specific metabolism of ferulic acid and resorcinol, suggesting that NlGSTs3 may be a promising target for RNAi-mediated BPH management.

Herbivore-induced plant volatiles (HIPVs) are known to prime neighboring plants for enhanced defense, but the molecular basis for this phenomenon remains poorly understood, particularly in cotton. Here, we demonstrate for cotton plants that exposure to volatiles induced by the cotton bollworm ( Helicoverpa armigera Hübner; CBW), enhances resistance against subsequent CBW attack, as evidenced in both laboratory and semi-field trials. While HIPV exposure alone did not elicit direct defense activation, it primed the jasmonic acid (JA) signaling pathway, leading to accelerated induction of JA biosynthesis genes and elevated JA accumulation upon herbivory. That this primed resistance is JA-dependent, was confirmed by treating HIPV-exposed plants with JA biosynthesis inhibitors, which completely abolished the priming effects. We further found that HIPV-primed plants exhibited significantly higher accumulation of the key defensive metabolite gossypol following larval feeding. The role of gossypol-reliant defenses was confirmed by using a glandless cotton mutant ( gl 2 gl 2 gl 3 gl 3 ) deficient in gossypol and related terpenoid aldehydes. The combined results reveal that CBW-induced volatiles prime anti-herbivore resistance in cotton by potentiating the JA signaling pathway, which in turn enhances gossypol biosynthesis upon actual herbivore attack. This new insight into the physio-ecological mechanisms underlying airborne defense priming in cotton also highlights its potential application in sustainable pest management.

P-element-induced wimpy testis (PIWI) -interacting RNAs (piRNAs) are short non-coding RNAs crucial for silencing transposable elements (TEs) and maintaining genome stability, particularly in germ cells. While most research on piRNAs and PIWI proteins has traditionally focused on model organisms such as Drosophila, emerging studies are now extending to other insect orders. However, little is known about piRNAs in insects of the Hemiptera order. Here, we investigated the role of piRNAs in the brown planthopper (BPH), Nilaparvata lugens, a significant pest of rice that belongs to the Hemiptera order. We identified and characterized two PIWI subfamily protein genes in N. lugens: NlAgo3 (a homolog of Ago3) and NlBiwi (the BPH Piwi). Both proteins contain Piwi/Argonaute/Zwille (PAZ) and PIWI domains. Sequence alignment and phylogenetic analysis demonstrated that NlAgo3 and NlBiwi are conserved across other insect orders. NlAgo3 and NlBiwi are highly expressed in female adults and their ovaries in N. lugens. Importantly, knockdown of NlAgo3 and NlBiwi significantly impaired eggs laying, leading to female sterility, suggesting that these proteins play a crucial role in female reproduction in N. lugens. Subsequently, through small RNA sequencing, we characterized piRNAs in N. lugens and identified a length peak in the range of 26-28 nucleotides, with differences in piRNA abundance between females and males. Additionally, we observed that piRNA clusters were distributed across each chromosome, with a higher density on the sex chromosomes. Moreover, RNAi-mediated knockdown of NlAgo3 and NlBiwi severely impaired piRNA production in BPH, highlighting that these proteins are essential for piRNA biogenesis in N. lugens, consistent with findings in other species. Overall, this study provides valuable insights into the role of piRNAs in the reproductive biology of N. lugens, a major pest of rice. By elucidating the characteristics of piRNAs in this insect species, this research enhances our understanding of piRNA-mediated gene regulation in Hemiptera insects. These findings have potential implications for the development of novel strategies for pest control.

Male-specific lethal-3 (MSL3) is a component of the dosage compensation complex in Drosophila melanogaster, where its mutation leads to male-specific lethality. However, the function of MSL3 in hemipteran insects remains unclear. This study investigated the role of the MSL3 homolog in a major rice pest, the brown planthopper (Nilaparvata lugens). We cloned and characterized the gene NlMSL3 from N. lugens, which is 1467-bp long and encodes a protein of 488 amino acids. Phylogenetic analysis revealed that MSL3 is conserved across various insect orders, with high conservation in the chromo-barrel domain. Quantitative real-time polymerase chain reaction indicated differential expression levels of NlMSL3 between male and female insects during development, with the highest expression in the testes. RNA interference-mediated knockdown of NlMSL3 in N. lugens resulted in significant mortality in later instar nymphs and adults compared with the control group. In females, NlMSL3 knockdown impaired feeding behavior, leading to decreased body weight, notably reduced honeydew excretion, flat abdomens, decreased vitellogenin expression, and defective ovarian development. When dsNlMSL3-treated males were mated with control females, the number of eggs laid was similar to that laid by the females mated with control males; however, none of the eggs laid by the former hatched into nymphs. These results highlight the crucial role of NlMSL3 in the development and fecundity of N. lugens.

The Cleavage Stimulation Factor (CstF) complex, consisting of three subunits, is essential for the 3′ end processing of precursor messenger RNA (pre-mRNA). In mammals, this complex includes CstF50, CstF64, and CstF77, named according to their molecular weights, and these proteins are conserved across many organisms. However, the functional roles of the three CstF genes (NlCstF50, NlCstF64, and NlCstF77) in Nilaparvata lugens, a major rice pest, have not been fully explored. This study identified and characterized the sequences of these genes, with proteins encoded by NlCstF50, NlCstF64, and NlCstF77 consisting of 439, 419, and 732 amino acids, respectively. These proteins are conserved among various insect species. Spatio-temporal expression analysis revealed that these genes are expressed at all developmental stages and in various tissues, with peak levels in eggs and testes. RNA interference (RNAi) targeting one or all three NlCstF genes resulted in a reduction in gene expression by 68% to 90% at 72 h post-injection, indicating that multi-gene dsRNA can achieve similar silencing outcomes as single-gene dsRNA. Knocking down one or all three NlCstF genes caused significant lethal phenotypes and molting disruptions. Mortality rates increased from 62.5% (dsNlCstF50) to 95.4% (dsNlCstF(50+64+77)). Additionally, silencing these genes reduced the number of eggs laid per female and hatch rates. These results highlight the critical role of NlCstF genes in the development and reproduction of N. lugens, suggesting their potential as targets for RNAi-based pest control strategies.

IntroductionThe brown planthopper (BPH) poses a significant threat to rice production in Asia. The use of resistant rice varieties has been effective in managing this pest. However, the adaptability of BPH to resistant rice varieties has led to the emergence of virulent populations, such as biotype Y BPH. YHY15 rice, which carries the BPH resistance gene Bph15, exhibits notable resistance to biotype 1 BPH but is susceptible to biotype Y BPH. Limited information exists regarding how resistant rice plants defend against BPH populations with varying levels of virulence.MethodsIn this study, we integrated miRNA and mRNA expression profiling analyses to study the differential responses of YHY15 rice to both avirulent (biotype 1) and virulent (biotype Y) BPH.ResultsYHY15 rice demonstrated a rapid response to biotype Y BPH infestation, with significant transcriptional changes occurring within 6 hours. The biotype Y-responsive genes were notably enriched in photosynthetic processes. Accordingly, biotype Y BPH infestation induced more intense transcriptional responses, affecting miRNA expression, defenserelated metabolic pathways, phytohormone signaling, and multiple transcription factors. Additionally, callose deposition was enhanced in biotype Y BPH-infested rice seedlings.DiscussionThese findings provide comprehensive insights into the defense mechanisms of resistant rice plants against virulent BPH, and may potentially guide the development of insect-resistant rice varieties.

为探究蛋白酶抑制剂基因在水稻抵御植食性昆虫为害中的功能，该研究以水稻品种 ‘中花11’为材料，克隆了水稻丝氨酸蛋白酶抑制剂基因OCPI2 编码区序列，并通过生物 信息学软件对其序列特征进行了分析和系统发育树的构建，同时采用实时荧光定量PCR 技 术探究了该基因在植食性昆虫取食和植物激素诱导下的表达特征。结果表明：（1）水稻 OCPI2 基因编码区序列全长219 bp，编码72 个氨基酸，预测蛋白分子量为7.72 kDa，理论 等电点为5.21，不含信号肽，无跨膜结构。（2）OCPI2 蛋白与乌拉尔图小麦(Triticum urartu, EMS61613.1)同源蛋白亲缘关系较近。（3）OCPI2 基因具有1 个potato_inhibit 保守结构域， 属于丝氨酸蛋白酶抑制剂家族。（4）二化螟、褐飞虱取食、机械损伤以及水杨酸甲酯处理 均能诱导OCPI2 基因的表达，茉莉酸甲酯处理则持续抑制OCPI2 表达。以上研究结果表 明OCPI2 基因可能参与了水稻对植食性昆虫的诱导防御反应，为深入研究OCPI2 在水稻 抗虫防御反应中的功能提供理论依据。

Brown planthoppers (BPHs, Nilaparvata lugens Stål) are a major threat to rice cultivation in Asia, necessitating the development of pest-resistant varieties for effective management. However, the adaptability of BPHs has resulted in the development of virulent populations, such as biotype Y BPHs, which exhibit significant virulence against the rice variety YHY15 that harbors the resistance gene Bph15. The various response mechanisms of BPH populations to resistant rice varieties are critical yet underexplored. Via RNA sequencing, the present study identified distinct transcriptional profiles in avirulent (biotype 1) and virulent (biotype Y) BPH nymphs both before and after feeding on YHY15 rice. Our findings revealed differential expression patterns of gene clusters involved in protein synthesis, hydrolysis, fatty acid biosynthesis, metabolism, cuticle composition, and translocation. Further analysis elucidated changes in the expression of genes associated with longevity and structural components of cuticles, highlighting specific disruptions in both biotype 1 and biotype Y BPHs. Moreover, the two biotypes showed differences in the expression level of genes involved in ATP-binding cassette (ABC) transporters. A functional assessment of ABC transporter genes revealed a role of NlABCG14 in the honeydew production of biotype Y BPHs to YHY15 rice, without impacting their survival and developmental dynamics. These insights deepen our understanding of the mechanisms of virulent BPHs response to resistant rice varieties and highlight potential targets for improving pest management strategies.

The brown planthopper (Nilaparvata lugens) is a major pest threatening global rice production, significantly reducing yields annually. As N. lugens increasingly develops resistance to conventional control methods, such as chemical pesticides, there is an urgent need for innovative and sustainable pest management strategies. Cleavage and Polyadenylation Specificity Factor 30 (CPSF30) is a key protein involved in mRNA 3′ end processing, yet its function in N. lugens remains poorly understood. This study aims to elucidate the role of CPSF30 in the growth and development of N. lugens and evaluate its potential as a target for RNA interference (RNAi)-based pest control strategies. We cloned and characterized the cDNA sequence of NlCPSF30, which encodes a protein of 341 amino acids containing five CCCH zinc-finger domains and two CCHC zinc-knuckle domains. Sequence alignment revealed that NlCPSF30 is highly conserved among insect species, particularly in the zinc-finger domains essential for RNA binding and processing. Phylogenetic analysis showed that NlCPSF30 is closely related to CPSF30 proteins from other hemipteran species. Expression analysis indicated that NlCPSF30 is most highly expressed in the fat body and during the adult stage, with significantly higher expression in females than in males. RNAi-mediated silencing of NlCPSF30 in third-instar nymphs resulted in severe phenotypic abnormalities, including disrupted molting and increased mortality following injection of double-stranded RNA (dsRNA) targeting NlCPSF30. Moreover, it influenced the expression of genes associated with hormone regulation, namely NlHry, NlE93, and NlKr-h1. These results suggest that NlCPSF30 is integral to critical physiological processes, with its disruption leading to increased mortality. Our findings identify NlCPSF30 as an essential gene for N. lugens’ survival and a promising target for RNAi-based pest management strategies. This study provides a valuable molecular target and theoretical insights for developing RNAi-based control methods against N. lugens.

Resource partitioning is considered to be a prerequisite for coexisting species to evolve from competition to mutualism. This is uniquely different for two major pest insects of rice. These herbivores preferentially opt to coinfest the same host plants, and through plant-mediated mechanisms, cooperatively utilize these plants in a mutualistic manner.

Interactions between rice plants (Oryza sativa L.) and brown planthoppers (Nilaparvata lugens Stål, BPHs) are used as a model system to study the molecular mechanisms underlying plant-insect interactions. Small RNAs (sRNAs) regulate growth, development, immunity, and environmental responses in eukaryotic organisms, including plants and insects. Recent research suggests that sRNAs play significant roles in rice-BPH interactions by mediating post-transcriptional gene silencing. The focus of this review is to explore the roles of sRNAs in rice-BPH interactions and to highlight recent research progress in unraveling the mechanism of cross-kingdom RNA interference (ckRNAi) between host plants and insects and the application of ckRNAi in pest management of crops including rice. The research summarized here will aid in the development of safe and effective BPH control strategies.

Herbivore-induced plant volatiles (HIPVs) are known to be perceived by neighboring plants, resulting in induction or priming of chemical defenses. There is little information on the defense responses that are triggered by these plant-plant interactions, and the phenomenon has rarely been studied in rice. Using chemical and molecular analyses in combination with insect behavioral and performance experiments, we studied how volatiles emitted by rice plants infested by the striped stemborer (SSB) Chilo suppressalis affect defenses against this pest in conspecific plants. Compared with rice plants exposed to the volatiles from uninfested plants, plants exposed to SSB-induced volatiles showed enhanced direct and indirect resistance to SSB. When subjected to caterpillar damage, the HIPV-exposed plants showed increased expression of jasmonic acid (JA) signaling genes, resulting in JA accumulation and higher levels of defensive proteinase inhibitors. Moreover, plants exposed to SSB-induced volatiles emitted larger amounts of inducible volatiles and were more attractive to the parasitoid Cotesia chilonis. By unraveling the factors involved in HIPV-mediated defense priming in rice, we reveal a key defensive role for proteinase inhibitors. These findings pave the way for novel rice management strategies to enhance the plant's resistance to one of its most devastating pests.

植物和植食性昆虫在长期的协同进化中形成复杂的防御和反防御机制.为应对植食性昆虫的取食,植物进化出组成型和诱导型等防御策略.相对于植物的防御,植食性昆虫在利用和适应寄主植物过程中,也进化出复杂的反防御机制.植食性昆虫可以利用唾液中的效应子干扰或者抑制寄主植物的防御反应,依靠解毒酶体系分解代谢源自植物的有毒物质,通过携带共生微生物间接抑制植物的抗虫防御反应,借助水平基因转移从细菌和植物等供体中获得相关功能基因以提高自身适合度,利用或者操控寄主植物挥发物等多种方式实现对寄主植物的反防御.此外,一些植食性昆虫还可以通过逃避、选贮和产卵等方式来抑制植物防御反应.解析植食性昆虫对植物的反防御机制,有助于深入理解植物-植食性昆虫协同进化关系,并为制定害虫防治措施提供新的思路.

Societal Impact Statement Genetic engineering is used to introduce new genes into plants to obtain crops with novel traits. However, the unintended effects of genetically engineered (GE) crops on nontarget organisms—such as beneficial insects—are a topic of much concern. We evaluate the potential unintended effects of pollen from three GE maize and seven commercialized conventional maize lines on the ladybird beetle Propylea japonica, by combining omics approaches with feeding assays. Our results suggest that unintended changes caused by genetic engineering in maize pollen may not lead to biologically relevant effects on P. japonica. Meanwhile our study provides a useful strategy to assess the biological impacts of genetic engineering on nontarget organisms. Summary The potential effects caused by the inserted traits in genetically engineered (GE) plants on nontarget organisms (NTOs) have been well assessed. However, whether the process of genetic engineering itself causes unintended changes that go beyond the natural variation of the crop and further poses any biological effects to NTOs is still under debate. Here, we evaluated the potential unintended effects of pollen from three GE maize and seven commercialized conventional maize lines on the NTO Propylea japonica by combining omics approaches with feeding assays. The results showed that genetic breeding indeed brought somewhat differences at both proteome and metabolome levels in maize pollen, although such differences were far more common in conventionally crossbred plants. Feeding experiments indicated that the changes in proteins and metabolites caused by genetic breeding did not lead to unintended effects on the NTOs that go beyond those measured for the conventional crossbred lines. Together, our results suggest that the differences detected by omics experiments may not cause any biological relevant effects on NTOs and the combination of omics approaches and NTOs feeding assays provide a valid approach to assess the biological relevance of compositional effects caused by genetic breeding.

为探明褐飞虱交配次数和性选择的生殖行为特征,构建了褐飞虱生物型1和生物型Y的近交系,筛选了在不同近交系之间存在明显差异的9个SSR(Simple Sequence Repeats)分子标记用于亲子鉴定.在雌虫交配次数试验中,观察并分子鉴定到23头成功交配的雌虫,其中19头雌虫一生仅进行1次交配,4头雌虫一生进行了2次交配,故褐飞虱雌虫是以单次交配为主、2次交配为辅的混合型交配策略.在性选择分析中,首先,性别内选择试验观察并鉴定到了23头交配成功的雄虫,其中18头生物型Y雄虫选择与雌虫进行交配;其次,性别间选择试验观察并鉴定到了83％生物型Y雄虫选择与雌虫进行交配.结果表明:在性选择行为中,生物型Y雄虫具有更强的竞争优势.

Normally, when different species of herbivorous arthropods feed on the same plant this leads to fitness-reducing competition. We found this to be different for two of Asia's most destructive rice pests, the brown planthopper and the rice striped stem borer. Both insects directly and indirectly benefit from jointly attacking the same host plant. Double infestation improved host plant quality, particularly for the stemborer because the planthopper fully suppresses caterpillar-induced production of proteinase inhibitors. It also reduced the risk of egg parasitism, due to diminished parasitoid attraction. Females of both pests have adapted their oviposition behaviour accordingly. Their strong preference for plants infested by the other species even overrides their avoidance of plants already attacked by conspecifics. This cooperation between herbivores is telling of adaptations resulting from the evolution of plant-insect interactions, and points out mechanistic vulnerabilities that can be targeted to control these major pests.

Plants typically release large quantities of volatiles in response to herbivory by insects. This benefits the plants by, for instance, attracting the natural enemies of the herbivores. We show that the brown planthopper (BPH) has cleverly turned this around by exploiting herbivore-induced plant volatiles (HIPVs) that provide safe havens for its offspring. BPH females preferentially oviposit on rice plants already infested by the rice striped stem borer (SSB), which are avoided by the egg parasitoid Anagrus nilaparvatae, the most important natural enemy of BPH. Using synthetic versions of volatiles identified from plants infested by BPH and/or SSB, we demonstrate the role of HIPVs in these interactions. Moreover, greenhouse and field cage experiments confirm the adaptiveness of the BPH oviposition strategy, resulting in 80% lower parasitism rates of its eggs. Besides revealing a novel exploitation of HIPVs, these findings may lead to novel control strategies against an exceedingly important rice pest.

Interactions between plants and insect herbivores are important determinants of plant productivity in cultivated and natural agricultural fields. The rice leaf folder (Cnaphalocrocis medinalis) causes tremendous damage to rice production in Asian countries. However, little information is available about how rice plants defend themselves against this destructive pest at molecular and biochemical levels. Here, we observed the transcriptomic and metabolomic differences in rice leaves after 0, 1, 6, 12, and 24 h of being fed by C. medinalis using RNA sequencing and metabolome profiling. Transcriptional analyses showed that gene expression responds rapidly to leaf folder infestation, with the most significant transcriptional changes occurring within 6 h after the initiation of feeding. Metabolite abundance changed more slowly than gene expression. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses indicated that the rice transcriptional response to infestation involved genes encoding protein kinases, transcription factors, biosynthesis of secondary metabolites, photosynthesis, and phytohormone signaling. Moreover, the jasmonic acid-dependent signaling pathway triggered by leaf folder herbivory played a vital role in rice defense against this pest. Taken together, our results provide comprehensive insights into the defense system of rice to this species and may inform the development of insect-resistant rice varieties.

SUMMARYAdvancements in ‐omics techniques provide powerful tools to assess the potential effects in composition of a plant at the RNA, protein and metabolite levels. These technologies can thus be deployed to assess whether genetic engineering (GE) causes changes in plants that go beyond the changes introduced by conventional plant breeding. Here, we compare the extent of transcriptome and metabolome modification occurring in leaves of four GE rice lines expressing Bacillus thuringiensis genes developed by GE and seven rice lines developed by conventional cross‐breeding. The results showed that both types of crop breeding methods can bring changes at transcriptomic and metabolic levels, but the differences were comparable between the two methods, and were less than those between conventional non‐GE lines were. Metabolome profiling analysis found several new metabolites in GE rice lines when compared with the closest non‐GE parental lines, but these compounds were also found in several of the conventionally bred rice lines. Functional analyses suggest that the differentially expressed genes and metabolites caused by both GE and conventional cross‐breeding do not involve detrimental metabolic pathways. The study successfully employed RNA‐sequencing and high‐performance liquid chromatography mass spectrometry technology to assess the unintended changes in new rice varieties, and the results suggest that GE does not cause unintended effects that go beyond conventional cross‐breeding in rice.