Jasmonate-mediated polyamine oxidase 6 drives herbivore-induced polyamine catabolism in rice.

Polyamines (PAs) along with their conjugated forms, are important mediators of plant defense mechanisms against both biotic and abiotic stresses. Flavin-containing polyamine oxidases (PAOs) regulate PA levels through terminal oxidation. To date, the role of PAOs in plant-herbivore interaction remains poorly understood. We discovered that infestation by the brown planthopper (BPH) disrupts PA homeostasis within the leaf sheaths of rice plants, which co-occurs with the upregulation of OsPAO6, a tissue-specific inducible, apoplast-localized enzyme that regulates the terminal catabolism of spermidine (Spd) and spermine. Functional analysis using CRISPR-Cas9 genome-edited plants revealed that pao6 mutants accumulated significantly higher levels of Spd and phenylpropanoid-conjugated Spd in response to BPH infestation compared to wild-type controls. In addition, BPH feeding on pao6 mutants led to increased honeydew excretion and plant damage by female adults, consistent with in vitro experiments in which Spd enhanced BPH feeding. Furthermore, OsPAO6 transcription is regulated by jasmonate (JA) signaling, and it is dependent on MYC2, which directly binds to the G-box-like motif in the OsPAO6 promoter. Our findings reveal an important role of OsPAO6 in regulating polyamine catabolism in JA-induced responses triggered by herbivore attacks in rice.

Identification and knockout of a herbivore susceptibility gene enhances planthopper resistance and increases rice yield.

Brown planthoppers (Nilaparvata lugens) and white-backed planthoppers (Sogatella furcifera) are among the most destructive pests on rice. However, plant susceptibility genes have not yet been exploited for crop protection. Here we identified a leucine-rich repeat protein, OsLRR2, from susceptible rice varieties that facilitates infestation by brown planthopper N. lugens. Field trials showed that knockout of OsLRR2 significantly reduced BPH infestation and enhanced natural biological control by attracting natural enemies. Yield of a susceptible variety was increased by 18% in insecticide-treated plots that eliminated planthoppers and by 25% in untreated plots. These findings underscore the pivotal role of OsLRR2, offering a promising pathway for pest population suppression and rice yield increase.

The jasmonate pathway and water loss in rice leaves induced by a stem-borer inhibit leaf-feeder growth.

Plant-mediated interactions between herbivores play an important role in regulating the composition of herbivore community. The fall armyworm (FAW), Spodoptera frugiperda, which has become one of the most serious pests on corn in China since it invaded in 2018, has been found feeding rice in the field. However, how FAW interacts with native rice insect pests remains largely unknown. Here, we investigated the interaction between FAW and a resident herbivore, striped stem borer (SSB, Chilo suppressalis) on rice. The infestation of rice leaf sheaths (LSs) by SSB larvae systemically enhanced the level of jasmonic acid (JA), abscisic acid (ABA), and trypsin proteinase inhibitors (TPIs), reduced relative water content (RWC) in leaf blades (LBs), and suppressed the growth of FAW larvae. In contrast, because FAW larvae infested LBs and did not affect defence responses in LSs, they did not influence the performance of SSB larvae. Using different mutants, together with bioassays and chemical analysis, we revealed that SSB-induced suppression of FAW larvae growth depended on both the SSB-activated JA pathway and RWC in LBs, whereas the ABA pathway activated by SSB larvae promoted the growth of FAW larvae by impeding water loss. These results provide new insights into mechanisms underlying plant-mediated interactions between herbivores.

OsRCI-1-Mediated GLVs Enhance Rice Resistance to Brown Planthoppers.

Green leaf volatiles (GLVs) play pivotal roles in plant anti-herbivore defense. This study investigated whether the rice 13-lipoxygense gene OsRCI-1 is involved in GLV production and plant defense in rice. The overexpression of OsRCI-1 (oeRCI lines) in rice resulted in increased wound-induced levels of two prominent GLVs, cis-3-hexen-1-ol and cis-3-hexenal. In a previous study, we found that the overexpression of OsRCI-1 reduced the colonization by the rice brown planthopper (BPH, Nilaparvata lugens) but increased the attractiveness to the egg parasitoid Anagrus nilaparvatae compared to wild-type (WT) plants. This study found that when cis-3-hexen-1-ol, but not cis-3-hexenal, was added to WT plants, it could change the BPH's colonization preference, i.e., more BPHs preferred to colonize the oeRCI lines. The exogenous application of cis-3-hexen-1-ol or cis-3-hexenal to BPH-infested WT plants could weaken or overturn the preference of A. nilaparvatae for oeRCI lines. However, field experiments revealed that only cis-3-hexenal was attractive to the parasitoid and increased the parasitism rates of BPH eggs. These results indicate that OsRCI-1 is involved in rice GLV production and therefore modulates both direct and indirect defense in rice.

Brown planthoppers manipulate rice sugar transporters to benefit their own feeding.

The feeding of piercing-sucking insect herbivores often elicits changes in their host plants that benefit the insect.1 In addition to thwarting a host's defense responses, these phloem-feeding insects may manipulate source-sink signaling so as to increase resources consumed.2,3 To date, the molecular mechanisms underlying herbivore-induced resource reallocation remain less investigated. Brown planthopper (BPH), an important rice pest, feeds on the phloem and oviposits into leaf sheaths. BPH herbivory increases sugar accumulations 5-fold in the phloem sap of leaf sheaths and concurrently induces the expression of two clade III SWEET genes, SWEET13 and SWEET14, in leaf tissues, but not in leaf sheaths of attacked rice plants. Mutations of both genes by genome editing attenuate resistance to BPH without alterations of known chemical and physical defense responses. Moreover, BPH-elicited sugar levels in the phloem sap were significantly reduced in sweet13/14 mutants, which is likely to attenuate BPH feeding behavior on sweet13/14 mutants. In one of the two field seasons tested, the sweet13/14 mutants showed comparable yield to wild types, and in the other season, the mutants demonstrated stronger BPH resistance. These preliminary results suggested that the mutations in these SWEET transporters could enhance BPH resistance without yield penalties. Given that sweet13/14 mutants also exhibit resistance to bacterial blight pathogen, Xanthomonas oryzae pv. oryzae, these SWEET genes could serve as excellent molecular targets for the breeding of resistant rice cultivars.

Multiomic analyses reveal key sectors of jasmonate-mediated defense responses in rice.

The phytohormone jasmonate (JA) plays a central role in plant defenses against biotic stressors. However, our knowledge of the JA signaling pathway in rice (Oryza sativa) remains incomplete. Here, we integrated multiomic data from three tissues to characterize the functional modules involved in organizing JA-responsive genes. In the core regulatory sector, MYC2 transcription factor transcriptional cascades are conserved in different species but with distinct regulators (e.g. bHLH6 in rice), in which genes are early expressed across all tissues. In the feedback sector, MYC2 also regulates the expression of JA repressor and catabolic genes, providing negative feedback that truncates the duration of JA responses. For example, the MYC2-regulated NAC (NAM, ATAF1/2, and CUC2) transcription factor genes NAC1, NAC3, and NAC4 encode proteins that repress JA signaling and herbivore resistance. In the tissue-specific sector, many late-expressed genes are associated with the biosynthesis of specialized metabolites that mediate particular defensive functions. For example, the terpene synthase gene TPS35 is specifically induced in the leaf sheath and TPS35 functions in defense against oviposition by brown planthoppers and the attraction of this herbivore's natural enemies. Thus, by characterizing core, tissue-specific, and feedback sectors of JA-elicited defense responses, this work provides a valuable resource for future discoveries of key JA components in this important crop.

Silencing an ATP-Dependent Caseinolytic Protease Proteolytic Subunit Gene Enhances the Resistance of Rice to Nilaparvata lugens.

The ATP-dependent caseinolytic protease (Clp) system has been reported to play an important role in plant growth, development, and defense against pathogens. However, whether the Clp system is involved in plant defense against herbivores remains largely unclear. We explore the role of the Clp system in rice defenses against brown planthopper (BPH) Nilaparvata lugens by combining chemical analysis, transcriptome, and molecular analyses, as well as insect bioassays. We found the expression of a rice Clp proteolytic subunit gene, OsClpP6, was suppressed by infestation of BPH gravid females and mechanical wounding. Silencing OsClpP6 enhanced the level of BPH-induced jasmonic acid (JA), JA-isoleucine (JA-Ile), and ABA, which in turn promoted the production of BPH-elicited rice volatiles and increased the resistance of rice to BPH. Field trials showed that silencing OsClpP6 decreased the population densities of BPH and WBPH. We also observed that silencing OsClpP6 decreased chlorophyll content in rice leaves at early developmental stages and impaired rice root growth and seed setting rate. These findings demonstrate that an OsClpP6-mediated Clp system in rice was involved in plant growth-defense trade-offs by affecting the biosynthesis of defense-related signaling molecules in chloroplasts. Moreover, rice plants, after recognizing BPH infestation, can enhance rice resistance to BPH by decreasing the Clp system activity. The work might provide a new way to breed rice varieties that are resistant to herbivores.

The N-terminal α2 helix element is critical for the activity of the rice transcription factor MYC2.

Jasmonates (JAs) are a class of phytohormones that play a crucial role in plant growth, development, and environmental stress responses. Central to JA signaling are the MYC2-type transcription factors, as they activate the expression of JA-responsive genes. We previously used CRISPR-Cas9-based genome editing to engineer rice OsMYC2 and yielded a mutant (myc2-5) with a single amino acid (aa) deletion (75I) outside the known functional domains of the protein. This myc2-5 mutant also showed some JA-deficient phenotypes, promoting us to investigate how 75I deletion affects JA responses. The mutation is found in the α2 helix element at the N-terminal of OsMYC2. The deletion of 75I in OsMYC2 rendered plants deficient in most of the JA responses, including root growth, leaf senescence, spikelet development, and resistance to pathogens and herbivores. Biochemical assays revealed that the 75I deletion markedly reduced OsMYC2 protein accumulation, subsequently diminishing its transcriptional activity. However, the deletion did not influence the protein's subcellular localization, DNA-binding capability, or its interactions with JAZ transcriptional repressors and the Mediator complex subunit MED25. Additionally, the screening of seven other deletions in the α2 helix further reinforces the importance of this protein element. Our results highlight the significance of the α2 helix in the N-terminus for OsMYC2's functionality, primarily through modulating its protein levels. This insight expands our knowledge of JA signaling and opens new avenues for research into the yet-to-be-explored domains of the MYC2 protein, with the potential to tailor JA responses in rice and other plant species.

Four-Chlorophenoxyacetic Acid Treatment Induces the Defense Resistance of Rice to White-Backed Planthopper Sogatella furcifera.

Chemical elicitors can increase plant defense against herbivorous insects and pathogens. The use of synthetic chemical elicitors is likely to be an alternative to traditional pesticides for crop pest control. However, only a few synthetic chemicals are reported to protect plants by regulating signaling pathways, increasing the levels of defense metabolites and interfering with insect feeding. Here, we found that the exogenous application of a phenoxycarboxylic compound, 4-chlorophenoxyacetic acid (4-CPA), can induce chemical defenses to protect rice plants from white-backed planthoppers (WBPH, Sogatella furcifera). Four-CPA was rapidly taken up by plant roots and degraded to 4-chlorophenol (4-CP). Four-CPA treatment modulated the activity of peroxidase (POD) and directly induced the deposition of lignin-like polymers using hydrogen peroxide (H2O2) as the electron acceptor. The polymers, which are thought to prevent the planthopper's stylet from reaching the phloem, were broken down by WBPH nymphs. Meanwhile, 4-CPA increased the levels of flavonoids and phenolamines (PAs). The increased flavonoids and PAs, together with the degradation product of the polymers, avoided nymphal feeding and prolonged the nymphal period for 1 day. These results indicate that 4-CPA has the potential to be used as a chemical elicitor to protect rice from planthoppers. Moreover, these findings also open a pathway for molecule structure design of phenoxycarboxylic compounds as chemical elicitors.

Knocking Out OsRLK7-1 Impairs Rice Growth and Development but Enhances Its Resistance to Planthoppers.

Leucine-rich repeat receptor-like kinases (LRR-RLKs) are an important subfamily of receptor-like kinases (RLKs) in plants that play key roles in sensing different biotic and abiotic stress. However, the role of LRR-RLKs in herbivore-induced plant defense remains largely elusive. Here, we found that the expression of a rice gene, OsRLK7-1, was induced by mechanical wounding, but was slightly suppressed by the infestation of gravid females of brown planthopper (BPH, Nilaparvata lugens) or white-backed planthopper (WBPH, Sogatella furcifera). Through targeted disruption of OsRLK7-1 (resulting in the ko-rlk lines), we observed an augmentation in transcript levels of BPH-induced OsMPK3, OsWRKY30, OsWRKY33, and OsWRKY45, alongside heightened levels of planthopper-induced jasmonic acid, JA-isoleucine, and abscisic acid in plant tissues. These dynamic changes further facilitated the biosynthesis of multiple phenolamides within the rice plants, culminating in an enhanced resistance to planthopper infestations under both lab and field conditions. In addition, knocking out OsRLK7-1 impaired plant growth and reproduction. These results suggest that OsRLK7-1 plays an important role in regulating rice growth, development, and rice-planthopper interactions.

Jasmonate-mediated gibberellin catabolism constrains growth during herbivore attack in rice.

Plant defense against herbivores is costly and often associated with growth repression. The phytohormone jasmonate (JA) plays a central role in prioritizing defense over growth during herbivore attack, but the underlying mechanisms remain unclear. When brown planthoppers (BPH, Nilaparvata lugens) attack rice (Oryza sativa), growth is dramatically suppressed. BPH infestation also increases inactive gibberellin (GA) levels and transcripts of GA 2-oxidase (GA2ox) genes, 2 (GA2ox3 and GA2ox7) of which encode enzymes that catalyze the conversion of bioactive GAs to inactive GAs in vitro and in vivo. Mutation of these GA2oxs diminishes BPH-elicited growth restriction without affecting BPH resistance. Phytohormone profiling and transcriptome analyses revealed that GA2ox-mediated GA catabolism was enhanced by JA signaling. The transcript levels of GA2ox3 and GA2ox7 were significantly attenuated under BPH attack in JA biosynthesis (allene oxide cyclase [aoc]) or signaling-deficient (myc2) mutants. In contrast, GA2ox3 and GA2ox7 expression was increased in MYC2 overexpression lines. MYC2 directly binds to the G-boxes in the promoters of both GA2ox genes to regulate their expression. We conclude that JA signaling simultaneously activates defense responses and GA catabolism to rapidly optimize resource allocation in attacked plants and provides a mechanism for phytohormone crosstalk.

Infestation by the Piercing-Sucking Herbivore Nilaparvata lugens Systemically Triggers JA- and SA-Dependent Defense Responses in Rice.

It has been well documented that an infestation of the piercing-sucking herbivore, brown planthopper (BPH), Nilaparvata lugens, activates strong local defenses in rice. However, whether a BPH infestation elicits systemic responses in rice remains largely unknown. In this study, we investigated BPH-induced systemic defenses by detecting the change in expression levels of 12 JA- and/or SA-signaling-responsive marker genes in different rice tissues upon a BPH attack. We found that an infestation of gravid BPH females on rice leaf sheaths significantly increased the local transcript level of all 12 marker genes tested except OsVSP, whose expression was induced only weakly at a later stage of the BPH infestation. Moreover, an infestation of gravid BPH females also systemically up-regulated the transcription levels of three JA-signaling-responsive genes (OsJAZ8, OsJAMyb, and OsPR3), one SA-signaling-responsive gene (OsWRKY62), and two JA- and SA- signaling-responsive genes (OsPR1a and OsPR10a). Our results demonstrate that an infestation of gravid BPH females systemically activates JA- and SA-dependent defenses in rice, which may in turn influence the composition and structure of the community in the rice ecosystem.

The Fall Armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae), Influences Nilaparvata lugens Population Growth Directly, by Preying on Its Eggs, and Indirectly, by Inducing Defenses in Rice.

The fall armyworm (FAW), Spodoptera frugiperda, has become one of the most important pests on corn in China since it invaded in 2019. Although FAW has not been reported to cause widespread damage to rice plants in China, it has been sporadically found feeding in the field. If FAW infests rice in China, the fitness of other insect pests on rice may be influenced. However, how FAW and other insect pests on rice interact remains unknown. In this study, we found that the infestation of FAW larvae on rice plants prolonged the developmental duration of the brown planthopper (BPH, Nilaparvata lugens (Stål)) eggs and plants damaged by gravid BPH females did not induce defenses that influenced the growth of FAW larvae. Moreover, co-infestation by FAW larvae on rice plants did not influence the attractiveness of volatiles emitted from BPH-infested plants to Anagrus nilaparvatae, an egg parasitoid of rice planthoppers. FAW larvae were able to prey on BPH eggs laid on rice plants and grew faster compared to those larvae that lacked available eggs. Studies revealed that the delay in the development of BPH eggs on FAW-infested plants was probably related to the increase in levels of jasmonoyl-isoleucine, abscisic acid and the defensive compounds in the rice leaf sheaths on which BPH eggs were laid. These findings indicate that, if FAW invades rice plants in China, the population density of BPH may be decreased by intraguild predation and induced plant defenses, whereas the population density of FAW may be increased.

Sakuranetin protects rice from brown planthopper attack by depleting its beneficial endosymbionts.

Plants produce chemical defenses that poison insect herbivores or deter their feeding, but herbivores are also accompanied by microbial endosymbionts crucial for their nutrition, reproduction, and fitness. Hence, plant defenses could target a herbivore's beneficial endosymbionts, but this has not yet been demonstrated. Here, we studied flavonoids that are induced when rice is attacked by a phloem-feeding pest, the brown planthopper (BPH), which harbors beneficial yeast-like symbionts (YLS) essential for insect nutrition, such as by remedying deficiencies in sterols. BPH attack dramatically increased sakuranetin accumulations in leaf sheaths and phloem exudates. Sakuranetin is an antifungal phytoalexin derived from the antibacterial precursor, naringenin, via catalysis of naringenin-O-methyltransferase (NOMT). When added to artificial diets, sakuranetin decreased BPH survivorship, suggesting that it functions as an induced defense. Mutation of NOMT abolished sakuranetin accumulation and increased BPH oviposition and hatching rates. High-throughput amplicon sequencing revealed that BPH fed on sakuranetin-deficient nomt lines were enriched in YLS with only minor changes in the bacterial endosymbionts, compared to those feeding on sakuranetin-rich wild-type (WT) plants. In-vitro feeding of sakuranetin suggested that this flavonoid directly inhibited the growth of YLS. BPH feeding on nomt lines accumulated higher cholesterol levels, which might be attributed to increases in the supply of sterol precursors from the YLS, while nomt lines suffered more damage than WT plants did from BPH herbivory. BPH-elicited accumulation of sakuranetin requires intact jasmonate (JA) signaling. This study reveals that rice uses a JA-induced antifungal flavonoid phytoalexin in defense against BPH by inhibiting its beneficial endosymbionts.

Functional dissection of rice jasmonate receptors involved in development and defense.

The phytohormones, jasmonates (JAs), mediate many plant developmental processes and their responses to important environmental stresses, such as herbivore attack. Bioactive JAs are perceived by CORONATINE INSENSITIVE (COI)-receptors, and associated JAZ proteins, to activate downstream responses. To date, the JA receptors of the important monocot crop plant, rice, remain to be explored. Here, we studied all three rice COI proteins, OsCOI1a, OsCOI1b, and OsCOI2, by ligand binding, genome editing, and phenotyping and examining some of the responsible mechanisms for the different responses. OsCOI2 binds to most individual OsJAZs in the presence of endogenous JA ligands, as OsCOI1a /1b do, albeit with greater partner selectivity. Single mutants of each OsCOI and OsCOI1a/1b double mutants were constructed by CRIPSR-Cas9-based genome editing and used to phenotype developmental and defense responses. OsCOI1b is involved in root growth and grain-size control and plays overlapping roles with OsCOI1a in spikelet development, while OsCOI2 regulates leaf senescence, male sterility, root growth, and grain size. All OsCOIs mediated resistance to the devastating rice pest, the brown planthopper. However, the defense sectors regulated by OsCOI1a/1b and OsCOI2 clearly differed. Our results revealed that all three OsCOIs are functional JA receptors that play diverse roles in regulating downstream JA responses.

The N-terminal subunit of vitellogenin in planthopper eggs and saliva acts as a reliable elicitor that induces defenses in rice.

Vitellogenins (Vgs) are critical for the development and fecundity of insects. As such, these essential proteins can be used by plants to reliably sense the presence of insects. We addressed this with a combination of molecular and chemical analyses, genetic transformation, bioactivity tests, and insect performance assays. The small N-terminal subunit of Vgs of the planthopper Nilaparvata lugens (NlVgN) was found to trigger strong defense responses in rice when it enters the plants during feeding or oviposition by the insect. The defenses induced by NlVgN not only decreased the hatching rate of N. lugens eggs, but also induced volatile emissions in plants, which rendered them attractive to a common egg parasitoid. VgN of other planthoppers triggered the same defenses in rice. We further show that VgN deposited during planthopper feeding compared with during oviposition induces a somewhat different response, probably to target the appropriate developmental stage of the insect. We also confirm that NlVgN is essential for planthopper growth, development, and fecundity. This study demonstrates that VgN in planthopper eggs and saliva acts as a reliable and unavoidable elicitor of plant defenses. Its importance for insect performance precludes evolutionary adaptions to prevent detection by rice plants.

Silencing a dehydration-responsive element-binding gene enhances the resistance of plants to a phloem-feeding herbivore.

Herbivore-induced plant defence responses share common components with plant responses to abiotic stresses. However, whether abiotic stress-responsive factors influence the resistance of plants to herbivores by regulating these components remains largely unknown. Here, we cloned a dehydration-responsive element-binding gene in rice, OsDREB1A, and investigated its role in the resistance of rice to the phloem-feeding herbivore, brown planthopper (BPH, Nilaparvata lugens), under normal and low temperatures. We found that OsDREB1A localized to the nucleus, and its transcripts in rice were up-regulated in response to BPH infestation, low temperatures and treatment with methyl jasmonate or salicylic acid. Silencing OsDREB1A changed transcript levels of two defence-related WRKY and two PLD genes, enhanced levels of jasmonic acid (JA), JA-isoleucine and abscisic acid, and decreased the ethylene level in rice; these changes subsequently enhanced the resistance of plants to BPH, especially at 17°C, by decreasing the hatching rate and delaying the development of BPH eggs. Moreover, silencing OsDREB1A increased the growth of rice plants. These findings suggest that OsDREB1A, which positively regulates the resistance of rice to abiotic stresses, negatively regulates the resistance of rice to BPH.

(-)-Loliolide is a general signal of plant stress that activates jasmonate-related responses.

The production of defensive metabolites in plants can be induced by signaling chemicals released by neighboring plants. Induction is mainly known from volatile aboveground signals, with belowground signals and their underlying mechanisms largely unknown. We demonstrate that (-)-loliolide triggers defensive metabolite responses to competitors, herbivores, and pathogens in seven plant species. We further explore the transcriptional responses of defensive pathways to verify the signaling role of (-)-loliolide in wheat and rice models with well-known defensive metabolites and gene systems. In response to biotic and abiotic stressors, (-)-loliolide is produced and secreted by roots. This, in turn, induces the production of defensive compounds including phenolic acids, flavonoids, terpenoids, alkaloids, benzoxazinoids, and cyanogenic glycosides, regardless of plant species. (-)-Loliolide also triggers the expression of defense-related genes, accompanied by an increase in the concentration of jasmonic acid and hydrogen peroxide (H2 O2 ). Transcriptome profiling and inhibitor incubation indicate that (-)-loliolide-induced defense responses are regulated through pathways mediated by jasmonic acid, H2 O2 , and Ca 2+ . These findings argue that (-)-loliolide functions as a common belowground signal mediating chemical defense in plants. Such perception-dependent plant chemical defenses will yield critical insights into belowground signaling interactions.

Characterization of Indoxacarb Resistance in the Fall Armyworm: Selection, Inheritance, Cross-Resistance, Possible Biochemical Mechanisms, and Fitness Costs.

The fall armyworm, Spodoptera frugiperda (J.E. Smith), is a voracious insect pest that is difficult to control due to resistance to insecticides and Bt proteins. We assessed cross-resistance, resistance mechanism, and fitness costs based on the life history traits of S. frugiperda. We established an S. frugiperda strain selected for resistance to indoxacarb (Ind-SEL) from a field-collected population and an unselected strain, Ind-UNSEL. Results indicated that after 24 generations of selection, the resistance to indoxacarb was increased by 472.67-fold as compared to the Ind-UNSEL. There was high cross-resistance to deltamethrin (31.23-fold) with very low or negligible cross-resistance to chlorantraniliprole, emamectin benzoate, and/or methoxyfenozide in the Ind-SEL population. Butoxide synergist increased susceptibility to indoxacarb, indicating that P450 enzymes may be involved in indoxacarb resistance. Significantly longer developmental time of larvae extended pupal duration, shorter adult longevity, and lower fecundity were observed in Ind-SEL as compared with the Ind-UNSEL population. The Net reproductive rate (R0) was the only growth parameter that differs between crosses of Ind-SEL♂ × Ind-UNSEL♀ (176 ± 46) and Ind-SEL♀ × Ind-UNSEL♂ (328 ± 57). On the other hand, all population growth parameters differ between Ind-SEL and Ind-UNSEL strains. Our work contributes to the growing body of research that demonstrates the importance of strain genetics in fitness cost experiments and helps resistance management programs make decisions.

Down-Regulation of P450 Genes Enhances Susceptibility to Indoxacarb and Alters Physiology and Development of Fall Armyworm, Spodoptera frugipreda (Lepidoptera: Noctuidae).

The fall armyworm (FAW), Spodoptera frugiperda (J.E. Smith), is a pest of many important crops globally. Effective control is challenging, with the pest exhibiting resistance to different synthetic pesticides across various groups. However, the mechanisms employed by resistant insects for overexpression of relevant detoxification genes remain unclear. The activity of detoxification enzymes was investigated in this study. Additionally, using RNA interference (RNAi), a functional analysis was completed of two P450s genes in an indoxacarb resistant population of fall armyworms. Elevated resistance levels (resistance ratio = 31.37-fold) in indoxacarb-selected populations of FAW were observed after 14 generations. The qRT-PCR showed higher expression of two cytochrome P450 genes, CYP321A7 and CYP6AE43, in this selected population compared to the control population. RNAi was applied to knock down the P450 dsCYP321A7 and dsCYP6AE43 genes in the FAW larvae. Droplet feeding of the dsRNAs (CYP321A7 and CYP6AE43) via an artificial diet significantly increased mortality rates in the indoxacarb treated population. A shorter larval developmental time of FAW was detected in all dsRNAs-fed larvae. Correspondingly, larval mass was reduced by dsRNAs in indoxacarb resistant populations of fall armyworm. Larval feeding assays demonstrate that dsRNAs targeting, specifically of CYP321A7 and CYP6AE43 enzymes, could be a beneficial technique in the management of indoxacarb resistant populations. Further study on the potential use of dsRNA and its application should be conducted in efforts to counter the development of resistance in FAW against various insecticides in the field.