Low concentrations of cyantraniliprole negatively affects the development of Spodoptera frugiperda by disruption of ecdysteroid biosynthesis and carbohydrate and lipid metabolism.

In addition to the acute lethal toxicity, insecticides might affect population dynamics of insect pests by inducing life history trait changes under low concentrations, however, the underlying mechanisms remain not well understood. Here we examined systemic impacts on development and reproduction caused by low concentration exposures to cyantraniliprole in the fall armyworm (FAW), Spodoptera frugiperda, and the putative underlying mechanisms were investigated. The results showed that exposure of third-instar larvae to LC10 and LC30 of cyantraniliprole significantly extended larvae duration by 1.46 and 5.41 days, respectively. Treatment with LC30 of cyantraniliprole significantly decreased the pupae weight and pupation rate as well as the longevity, fecundity and egg hatchability of female adults. Consistently, we found that exposure of FAW to LC30 cyantraniliprole downregulated the mRNA expression of four ecdysteroid biosynthesis genes including SfNobo, SfShd, SfSpo and SfDib and one ecdysone response gene SfE75 in the larvae as well as the gene encoding vitellogenin (SfVg) in the female adults. We also found that treatment with LC30 of cyantraniliprole significantly decreased the whole body levels of glucose, trehalose, glycogen and triglyceride in the larvae. Our results indicate that low concentration of cyantraniliprole inhibited FAW development by disruption of ecdysteroid biosynthesis as well as carbohydrate and lipid metabolism, which have applied implications for the control of FAW.

Functional Characterization and Putative Regulatory Mechanism of an RNAi Efficiency-Related Nuclease (REase) in the Fall Armyworm, Spodoptera frugiperda.

The lepidopteran-specific RNAi efficiency-related nuclease (REase) has been shown to contribute to double-strand RNA (dsRNA) degradation in several lepidopteran insects. However, little is known about its regulatory mechanism. In this study, we identified and characterized SfREase in Spodoptera frugiperda. The exposure of the third-instar larvae to dsEGFP and high temperature led to the upregulation of SfREase, whereas starvation treatment resulted in the downregulation of SfREase. Further experiments revealed that dsRNA degraded more slowly in the hemolymph or midgut fluid extracted from dsSfREase-injected or dsSfREase-ingested larvae compared with those from dsEGFP-treated larvae, and the recombinant SfREase degraded dsRNA in a concentration-dependent manner. Additionally, the knockdown of SfREase improved RNAi efficiency. Finally, both RNAi and dual-luciferase reporter assay in Sf9 cells revealed that SfREase is negatively regulated by FOXO. These data provide insights into the function and regulatory mechanism of REase and have applied implications for the development of an RNAi-based control strategy of S. frugiperda.

Involvement of Glutamate Cysteine Ligase Genes in Tolerance to Emamectin Benzoate in Spodoptera frugiperda and Their Putative Regulatory Mechanisms.

As the rate-limiting enzyme in de novo Glutathione (GSH) biosynthesis, the mammalian glutamate cysteine ligase (Gcl) catalytic (Gclc) and modifier (Gclm) subunits are regulated at multiple levels, whereas the function and regulatory mechanism of insect Gcl remain to be explored. In this study, we identified and characterized SfGclc and SfGclm in Spodoptera frugiperda. SfGclc and SfGclm were highly expressed in the hindgut and relatively less expressed in other tissues. The exposure of the third instar larvae to LC30 of emamectin benzoate (EMB) significantly reduced the GSH content with a concomitant upregulation of SfGclc and SfGclm. Further in vivo pretreatment with L-BSO, the Gcl inhibitor, increased the susceptibility of S. frugiperda to EMB. Consistently, overexpression of SfGclc and SfGclm increased the Sf9 cell viability under EMB treatment. Finally, both RNAi and the dual-luciferase reporter assay in Sf9 cells revealed that SfGclc is regulated by transcription factor CncC. These data provide insights into the function and regulatory mechanism of insect Gcl, and they imply that disruption of the redox homeostasis might be a practical strategy to enhance the insecticidal activity of EMB and other insecticides.

Restoration of energy homeostasis under oxidative stress: Duo synergistic AMPK pathways regulating arginine kinases.

Rapid depletion of cellular ATP can occur by oxidative stress induced by reactive oxygen species (ROS). Maintaining energy homeostasis requires the key molecular components AMP-activated protein kinase (AMPK) and arginine kinase (AK), an invertebrate orthologue of the mammalian creatine kinase (CK). Here, we deciphered two independent and synergistic pathways of AMPK acting on AK by using the beetle Tribolium castaneum as a model system. First, AMPK acts on transcriptional factor forkhead box O (FOXO) leading to phosphorylation and nuclear translocation of the FOXO. The phospho-FOXO directly promotes the expression of AK upon oxidative stress. Concomitantly, AMPK directly phosphorylates the AK to switch the direction of enzymatic catalysis for rapid production of ATP from the phosphoarginine-arginine pool. Further in vitro assays revealed that Sf9 cells expressing phospho-deficient AK mutants displayed the lower ATP/ADP ratio and cell viability under paraquat-induced oxidative stress conditions when compared with Sf9 cells expressing wild-type AKs. Additionally, the AMPK-FOXO-CK pathway is also involved in the restoration of ATP homeostasis under oxidative stress in mammalian HEK293 cells. Overall, we provide evidence that two distinct AMPK-AK pathways, transcriptional and post-translational regulations, are coherent responders to acute oxidative stresses and distinguished from classical AMPK-mediated long-term metabolic adaptations to energy challenge.

Mechanisms underlying the effects of low concentrations of chlorantraniliprole on development and reproduction of the fall armyworm, Spodoptera frugiperda.

It is well known that sublethal dose of insecticides induces life history trait changes of both target and non-target insect species, however, the underlying mechanisms remain not well understood. In this study, the effects of low concentrations of the anthranilic diamide insecticide chlorantraniliprole on the development and reproduction of the fall armyworm (FAW), Spodoptera frugiperda, were evaluated, and the underlying mechanisms were explored. The results showed that exposure of FAW to LC10 and LC30 chlorantraniliprole prolonged the larvae duration, decreased the mean weight of the larvae and pupae, and lowered the pupation rate as well as emergence rate. The fecundity of female adults was also negatively affected by treatment with low concentrations of chlorantraniliprole. Consistently, we found that exposure of FAW to LC30 chlorantraniliprole downregulated the mRNA expression of juvenile hormone (JH) esterase (SfJHE), leading to the increase of JH titer in larvae. We also found that treatment with low concentrations of chlorantraniliprole suppressed the expression of ribosomal protein S6 kinase1 (SfS6K1) in female adults, resulting in the downregulation of the gene encoding vitellogenin (SfVg). These results provided insights into the mechanisms underlying the effects of low concentrations of insecticides on insect pests, and had applied implications for the control of FAW.

Comparative phosphoproteomics analysis provides insights into the responses of Chilo suppressalis to sublethal chlorantraniliprole exposure.

BACKGROUND: Sublethal exposure to insecticides causes changes in insect behaviors and physiologies including feeding, mobility, communication, hormone homeostasis, development and fecundity, however, the underlying molecular mechanisms were largely unclear. Our previous studies revealed that sublethal chlorantraniliprole exposure disturbed the hormone homeostasis, reduced the weight and longevity and prolonged the developmental duration of Chilo suppressalis. In the present study, the potential phosphorylation modification regulation mechanisms in C. suppressalis in response to sublethal chlorantraniliprole exposure were explored using comparative and quantitative phosphoproteomics. RESULTS: A total of 2640 phosphopeptides belonging to 1144 phosphoproteins were identified, among which 446 phosphopeptides derived from 303 unique phosphoproteins were differentially phosphorylated between the chlorantraniliprole-treated and control larvae. The phosphorylation levels of differentially phosphorylated phosphopeptides were further validated using parallel reaction monitoring (PRM). Functional classification and protein-protein interaction of the differentially phosphorylated proteins (DPPs) were analyzed. Generalized analysis of the DPPs and the differentially expressed genes (DEGs) identified in our previous study showed that sublethal chlorantraniliprole exposure significantly changed the transcription and phosphorylation levels of genes/proteins associated with carbohydrate and lipid metabolism, cytoskeleton, signal transduction, transcription, translation and post-translational modification, leading to the dysfunctions of energy metabolism, transcription regulation, protein synthesis and modification, and signal transduction in C. suppressalis. Further analysis of the phosphorylation motifs in DPPs revealed that the MAPKs, CDKs, CaMK II, PKA, PKC and CK II protein kinases might be directly responsible for the phosphoproteomics response of C. suppressalis to chlorantraniliprole treatment. CONCLUSION: Our results provide abundant phosphorylation information for characterizing the protein modification in insects, and also provide valuable insights into the molecular mechanisms of insect post-translational modifications in response to sublethal insecticide exposure. © 2023 Society of Chemical Industry.

The pleiotropic AMPK-CncC signaling pathway regulates the trade-off between detoxification and reproduction.

The association of decreased fecundity with insecticide resistance and the negative sublethal effects of insecticides on insect reproduction indicates the typical trade-off between two highly energy-demanding processes, detoxification and reproduction. However, the underlying mechanisms are poorly understood. The energy sensor adenosine monophosphate-activated protein kinase (AMPK) and the transcription factor Cap "n" collar isoform C (CncC) are important regulators of energy metabolism and xenobiotic response, respectively. In this study, using the beetle Tribolium castaneum as a model organism, we found that deltamethrin-induced oxidative stress activated AMPK, which promoted the nuclear translocation of CncC through its phosphorylation. The CncC not only acts as a transcription activator of cytochrome P450 genes but also regulates the expression of genes coding for ecdysteroid biosynthesis and juvenile hormone (JH) degradation enzymes, resulting in increased ecdysteroid levels as well as decreased JH titer and vitellogenin (Vg) gene expression. These data show that in response to xenobiotic stress, the pleiotropic AMPK-CncC signaling pathway mediates the trade-off between detoxification and reproduction by up-regulating detoxification genes and disturbing hormonal homeostasis.

Molecular Characterization of Spodoptera frugiperda Heme Oxygenase and Its Involvement in Susceptibility to Chlorantraniliprole.

The mammalian heme oxygenase (HO) plays an important role in cytoprotection against oxidative-stress-induced cell damage; however, functional characterization of insect HO is still limited. In this study, cDNA encoding a HO, named SfHO, was cloned from Spodoptera frugiperda. Analysis of the transcription level and enzymatic activity showed that exposure of the LC30 concentration of chlorantraniliprole to the third instar larvae significantly upregulated both the mRNA level and enzymatic activity of SfHO at 24 h after treatment. Further injection of the HO activator, hemin, into the third instar larvae led to the upregulation of SfHO as well as decreased susceptibility of S. frugiperda to chlorantraniliprole. Consistently, overexpression of SfHO increased the Sf9 cell viability under chlorantraniliprole treatment. Strikingly, both RNAi and the dual-luciferase reporter assay in Sf9 cells revealed that, unlike mammalian HO that is regulated by the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), SfHO was not subject to the regulation by cap 'n' collar isoform C (CncC), the Nrf2 homologue in insects. These data provide insights into the function and regulatory mechanism of insect HOs and had applied implications for the control of S. frugiperda.

S6K1 acts through FOXO to regulate juvenile hormone biosynthesis in the red flour beetle, Tribolium castaneum.

As the downstream effector of the target of rapamycin complex 1 (TORC1) signaling pathway, the ribosomal protein S6 kinase (S6K) is an important regulator of insect reproduction, however, the underlying mechanism remains obscure. In this study, a S6K gene, named TcS6K1, was isolated from the red flour beetle, Tribolium castaneum. Analysis of temporal and spatial expression patterns revealed that TcS6K1 is expressed at the highest level in the one-day-old first instar larvae and head of 7-day-old females, respectively. RNAi-mediated knockdown of TcS6K1 in either female or male adults decreased the number of eggs laid, with a concomitant reduction of mRNA levelsof vitellogenin genes, TcVg1 and TcVg2, two male accessory gland secretory proteins, as well as the juvenile hormone (JH) biosynthesis-related gene, farnesol dehydrogenase (TcFDH). While the mRNA and protein levels of the transcription factor forkhead box O (TcFOXO) were not affected, suppression of TcS6K1 expression promoted TcFOXO nuclear translocation to exert its transcriptional action. Further RNAi and EMSA analysis revealed that TcFOXO negatively regulated the expression of TcFDH. These results indicate that S6K might regulate beetles' reproduction through FOXO/JH signaling pathway.

Knockdown or inhibition of arginine kinases enhances susceptibility of Tribolium castaneum to deltamethrin.

Metabolism of insecticides is an energy-consuming process. As an important component of the intracellular energy buffering system, arginine kinase (AK) plays an important role in insect cellular energy homeostasis and environmental stress response, but the involvement of AKs in the response to chemical stressors (insecticides) remains largely unknown. In this study, using Tribolium castaneum as a model organism, we found that deltamethrin treatment significantly upregulated the expression of TcAK1 and TcAK2 and decreased the whole body ATP content. The knockdown of TcAK1 or TcAK2 significantly enhances the deltamethrin-induced ATP depletion and increase the susceptibility of T. castaneum to deltamethrin. In addition, pretreatment with two AK inhibitors, rutin and quercetin, significantly decreased the lifespan of beetles treated with deltamethrin. These results suggest that AKs might be involved in detoxification of insecticides by regulating cellular energy balance.

FOXO acts as a positive regulator of CncC and deltamethrin tolerance in the red flour beetle, Tribolium castaneum.

BACKGROUND: Both forkhead box O (FOXO) and nuclear factor erythroid-derived 2-like-2 (Nrf2) are key transcription factors related to stress responses. Whereas limited studies in mammals and Caenorhabditis elegans have revealed the interaction between FoxO/DAF-16 and Nrf2/SKN-1, the role of FOXO in metabolic detoxification and regulation of the Nrf2-Keap1 signaling pathway are poorly understood in insects. RESULTS: Using Tribolium castaneum as a model organism, we found that RNA interference-mediated knockdown of FOXO enhanced deltamethrin-induced lethality by affecting the messenger RNA (mRNA) levels of CYP6BQ cluster genes. We further demonstrated that injection of dsFOXO into the beetle larvae decreased expression of CncC and KEAP1 at both the mRNA and protein level. Notably, dual-luciferase and electrophoretic mobility shift assays both confirmed direct regulation of CncC by FOXO, whereas Keap1 was directly regulated by CncC. CONCLUSION: FOXO can directly regulate the expression of CncC and indirectly regulate the expression of Keap1 through CncC. The data provide insights into the regulatory mechanisms of the Nrf2-Keap1 signaling pathway in insects.

The cytosolic sulfotransferase gene TcSULT1 is involved in deltamethrin tolerance and regulated by CncC in Tribolium castaneum.

The sulfuryl transfer reaction catalyzed by cytosolic sulfotransferase (SULT) is one of the major conjugating pathways responsible for the detoxification and subsequent elimination of xenobiotics, however, functional characterization of insect SULTs is still limited. In this study, cDNA encoding a cytosolic sulfotransferase, named TcSULT1, was cloned from the red flour beetle, Tribolium castaneum. Sequence analysis revealed that TcSULT1 had the conserved signature sequences of SULTs, and shared moderate amino acid identities with Bombyx mori and Drosophila SULTs. Analysis of the transcription level showed that TcSULT1 was highly expressed in head, epidermis and malpighian tube, and upregulated at 4 h after exposure to deltamethrin. Knockdown of TcSULT1 significantly increased the susceptibility of beetles to deltamethrin. Both RNAi and dual-luciferase assay revealed that the transcription factor TcCncC regulates the expression of TcSULT1. These data provides insights into the function and regulatory mechanism of insect SULTs.

20-hydroxyecdysone regulates expression of methioninesulfoxide reductases through transcription factor FOXO in the red flour beetle, Tribolium castaneum.

The oxidation of methionine (Met) by reactive oxygen species (ROS) causes detrimental effects on the protein functions. Methionine sulfoxide reductase (Msr) is the secondary antioxidant enzyme involved in protein repair, and is divided into two distinct classes, MsrA and MsrB, although the mechanisms underlying the transcriptional regulation of Msrs remain largely unknown. In this study, the full-length cDNAs encoding MsrA and three alternatively spliced isoforms of MsrB were isolated from the red flour beetle, Tribolium castaneum. Exposure of female adults to oxidative, heat and cold stresses induced expressions of both MsrA and MsrB. RNAi-mediated knockdown of MsrA and MsrB resulted in increased sensitivity of T. castaneum to paraquat-induced oxidative stress. Treatment with 20-hydroxyecdysone (20E) increased expression levels of both MsrA and MsrB. Knockdown of transcription factor forkhead box O (FOXO) decreased both MsrA and MsrB mRNA levels and abolished the induction of MsrA and MsrB by paraquat. Luciferase reporter assays revealed that FOXO directly activates the promoters of both MsrA and MsrB. Moreover, paraquat treatment induced expression of two ecdysone biosynthesis genes, Shade and Phantom, 20E upregulated exoression of FOXO, promoted FOXO nuclear translocation，and knockdown of FOXO abolished induction of MsrA and MsrB expression by 20E, suggesting that regulation of MsrA and MsrB by 20E was mediated by FOXO. Overall, these results provide important insights into the transcriptional regulation of insect Msrs.

Transcriptional and post-translational activation of AMPKα by oxidative, heat, and cold stresses in the red flour beetle, Tribolium castaneum.

The AMP-activated protein kinase (AMPK) has important roles in the regulation of energy metabolism, and AMPK activity and its regulation have been the focus of relevant investigations. However, functional characterization of AMPK is still limited in insects. In this study, the full-length cDNA coding AMPKα (TcAMPKα) was isolated from the red flour beetle, Tribolium castaneum. The TcAMPKα gene contains an ORF of 1581 bp encoding a protein of 526 amino acid residues, which shared conserved domain structure with Drosophila melanogaster and mammalian orthologs. Exposure of female adults to oxidative, heat, and cold stresses caused an increase in TcAMPKα mRNA expression levels and phosphorylation of Thr-173 in the activation loop. The RNAi-mediated knockdown of TcAMPKα resulted in the increased sensitivity of T. castaneum to oxidative, heat, and cold stresses. These results suggest that stress signals regulate TcAMPKα activity, and TcAMPKα plays an important role in enabling protective mechanisms and processes that confer resistance to environmental stress.

Transcriptome analysis reveals global gene expression changes of Chilo suppressalis in response to sublethal dose of chlorantraniliprole.

The anthranilic diamide insecticide chlorantraniliprole was widely used for the controlling of Chilo suppressalis in China. Previous studies have revealed sublethal effects of chlorantraniliprole on the development and reproduction of C. suppressalis. In the present study, a comparative transcriptome analysis was performed to investigate the global gene expression changes in third-instar larvae of C. suppressalis after exposure to LC30 of chlorantraniliprole. A total of 908 differentially expressed genes (DEGs) were identified including 441 up-regulated and 467 down-regulated unigenes. Gene enrichment analysis revealed that the down-regulated DEGs were mainly linked to carbohydrate, energy, lipid and amino acid metabolisms as well as posttranslational modification, while most of the DEGs involved in signal transduction were up-regulated. Specifically, the DEGs encoding detoxification related genes were identified and validated by RT-qPCR. Our results provide a basis for understanding the molecular mechanisms of chlorantraniliprole action and detoxification in C. suppressalis and other insect pests.

Molecular characterization of glutamate-gated chloride channel and its possible roles in development and abamectin susceptibility in the rice stem borer, Chilo suppressalis.

Glutamate-gated chloride channels (GluCls) mediate fast inhibitory neurotransmission in invertebrate nervous systems, and are of considerable interest in insecticide discovery. The full length cDNA encoding CsGluCl was cloned from the rice stem borer Chilo suppressalis (Walker). Multiple cDNA sequence alignment revealed three variants of CsGluCl generated by alternative splicing of exon 3 and exon 9. While all the transcripts were predominantly expressed in both nerve cord and brain, the expression patterns of these three variants differed among other tissues and developmental stages. Specifically, the expression level of CsGluCl C in cuticle was similar to that in nerve cord and brain, and was the predominant variant in late pupae and early adult stages. Both injection and oral delivery of dsGluCl significantly reduced the mRNA level of CsGluCl. Increased susceptibility to abamectin and reduced larvae growth and pupation rate were observed in dsGluCl-treated larvae. Thus, our results provide the evidence that in addition to act as the target of abamectin, GluCls also play important physiological roles in the development of insects.

Knockdown of the GABA receptor RDL genes decreases abamectin susceptibility in the rice stem borer, Chilo suppressalis.

The γ-aminobutyric acid (GABA) receptor is a primary neurotransmitter receptor in both vertebrate and invertebrate nervous systems. Multiple RDL subunits have been found in insects including the rice stem borer, Chilo suppressalis, however, comparative characterization of duplicated RDL genes in insects is still limited. In this study, comparison of the genomic sequences and the cDNA sequences revealed that both CsRDL1 and CsRDL2 consisted of 10 exons and 9 introns, and their exon-intron boundaries occur in the same position with respect to the coding sequences. Expression profiling showed that both CsRDL1 and CsRDL2 were predominantly expressed in nervous system, and had low expression levels in the gut and integument. The transcript level of CsRDL2 dramatically increased from the prepupae to late pupae and were much higher than that of CsRDL1 in adult stages. Notably, dietary ingestion of dsRDL1 and dsRDL2 significantly decreased the larval susceptibility to abamectin. These results suggest that CsRDL1 and CsRDL2 might play both physiological roles in development and toxicological roles in action of abamectin in C. suppressalis.