Knockdown of CYP9A9 increases the susceptibility to lufenuron, methoxyfenozide and a mixture of both in Spodoptera exigua.

Spodoptera exigua (Lepidoptera, Noctuidae) poses significant threats to global agriculture, necessitating the management of resistance through sustainable alternatives such as Insect growth regulators (IGRs), including Methoxyfenozide (MET) and Lufenuron (LUF). This study elucidates the potential regulatory mechanisms between 20-hydroxyecdysone (20E) and immune signaling under IGR exposure. Sublethal doses of MET, LUF, and their mixture (MML) significantly downregulated the 20E titer by upregulating SeShd and SeCYP18A1. Silencing SeShd decreased 20E, whereas knockdown SeCYP18A1 increased 20E; both delayed development and increased susceptibility to IGRs. Furthermore, IGRs activated immune pathways (Toll/IMD), elevating SeDif and SePGRP-LB (SePLB) expression while simultaneously suppressing 20E. Silencing SeDif and SePLB stimulated 20E biosynthesis and heightened susceptibility to IGRs. These findings offer novel insights into the intricate interplay between 20E biosynthesis and the immune response to IGRs. Our work enhances the understanding of the IGRs' molecular mechanism and offers actionable strategies for combating resistance in S. exigua.

In insects, cytochrome P450 monooxygenases (P450s or CYPs) are known to be involved in the detoxification and metabolism of insecticides, leading to increased resistance in insect populations. Spodoptera exigua is a serious polyphagous insect pest worldwide and has developed resistance to various insecticides. In this study, a novel CYP3 clan P450 gene CYP9A105 was identified and characterized from S. exigua. The cDNAs of CYP9A105 encoded 530 amino acid proteins, respectively. Quantitative real-time PCR analyses showed that CYP9A105 was expressed at all developmental stages, with maximal expression observed in fifth instar stage larvae, and in dissected fifth instar larvae the highest transcript levels were found in midguts and fat bodies. The expression of CYP9A105 in midguts was upregulated by treatments with the insecticides α-cypermethrin, deltamethrin and fenvalerate at both LC15 concentrations (0.10, 0.20 and 5.0 mg/L, respectively) and LC50 concentrations (0.25, 0.40 and 10.00 mg/L, respectively). RNA interference (RNAi) mediated silencing of CYP9A105 led to increased mortalities of insecticide-treated 4th instar S. exigua larvae. Our results suggest that CYP9A105 might play an important role in α-cypermethrin, deltamethrin and fenvalerate detoxification in S. exigua.

Knockdown of UDP-N-acetylglucosamine pyrophosphorylase and chitin synthase A increases the insecticidal efficiency of Lufenuron to Spodoptera exigua

Plants employ an intricate and dynamic defense system that includes physiological, biochemical, and molecular mechanisms to counteract the effects of herbivorous attacks. In addition to their tolerance to phytotoxins, beet armyworm has quickly developed resistance to deltamethrin; a widely used pyrethroid insecticide in cotton fields. The lethal concentration (LC50) required to kill 50% of the population of deltamethrin to gossypol-fed Spodoptera exigua larvae was 2.34-fold higher than the control group, suggesting a reduced sensitivity as a consequence of the gossypol diet. Piperonyl butoxide (PBO) treatment was found to synergize with deltamethrin in gossypol-fed S. exigua larvae. To counteract these defensive plant secondary metabolites, beet armyworm elevates their production of detoxification enzymes, including cytochrome P450 monooxygenases (P450s). Gossypol-fed beet armyworm larvae showed higher 7-ethoxycoumarin-O-deethylase (ECOD) activities and exhibited enhanced tolerance to deltamethrin after 48 and 72 h when compared to the control. Moreover, gossypol pretreated S. exigua larvae showed faster weight gain than the control group after transferring to a deltamethrin-supplemented diet. Meanwhile, gossypol-induced P450s exhibited high divergence in the expression level of two P450 genes: CYP6AB14 and CYP9A98 in the midgut and fat bodies contributed to beet armyworm tolerance to deltamethrin. Knocking down of CYP6AB14 and CYP9A98, via double-stranded RNAs (dsRNA) in a controlled diet, rendered the larvae more sensitive to the insecticide. These data demonstrate that generalist insects can exploit secondary metabolites from host plants to enhance their defense systems against other toxic chemicals. Impairing this defense pathway by RNA interference (RNAi) holds a potential to eliminate the pest’s tolerance to insecticides and, therefore, reduce the required dosages of agrochemicals in pest control.

High relative humidity is associated with high mortality in insects due to the infection with entomopathogens. However, the mechanism is still not fully understood. In this study, bioassay for Spodoptera exigua nucleopolhedrovirus (SeNPV) and antioxidant enzyme activity, total antioxidant capacity, and hydroxyl radical scavenging capacity were measured in Spodoptera exigua (Hübner) larvae under three relative humidities (RH) conditions (60–70 %, 75–85 %, and 90–100 %). Results showed that humidity significantly affected the toxicity of SeNPV against S. exigua larvae, and the LD50 (lethal dose 50 %) of SeNPV against S. exigua larvae was 1539.8 (60–70 % RH), 1110.4 (75–85 % RH) and 567.6 PIBs/g (90–100 % RH). The activities of superoxide dismutase (SOD), catalase (CAT), and peroxidase (POX) activities of S. exigua larvae were significantly lower in those subjected to high humidity than those in the control humidity group (60–70 % RH). The capacity of total antioxidant and hydroxyl radical scavenging decreased with the increasing of relative humidity. These results suggest that high RH may cause a decrease in the antioxidant system of S. exigua larvae, which may explain high RH is always associated with high mortality in insects due to infection with pathogens.

Neem-seed extract, incorporated into an artificial diet at 0.02, 0.2, and 2.0% (wt/vol) prolonged development and induced mortality in all larval stages of Trichoplusiani (Hubner) and Spodoptera exigua (Hubner). Only one nonreproductive T. ni adult female was produced at the lowest concentration (0.02%) when fifth instars were exposed to the diet. No pupae were formed by S. exigua larvae, regardless of stage treated or extract concentration tested. Larva mortality of both species was more pronounced during ecdysis, indicating activity similar to that of other insect growth regulators.

The aim of this study is to determine the Spodoptera exigua larva population in the field and factor affecting their density. Population study of S. exigua larva and its affecting factors was carried out in Sekinchan, Selangor during 2003-2004. The larval density was found fluctuating during the study, where the highest number of larvae was an averaged of 18.17 per m2, while the lowest number was an averaged of 1.5 per m2. The mean number of larvae per plant also varies from 1.83 to 5.42. It was found that the larval density was influenced by the age and availability of the host plant. A total of 1881 larvae were collected, where 18.29 and 20.31% were successfully becoming female and male moths, respectively; 20.63% was being parasitized, where 7.07, 11.43, 0.11 and 2.02% were being parasitized by Microplitis manilae, Chelonus sp., Temelucha sp. and Peribaea orbata, respectively. Besides that, other biotic factors such as fungal or bacterial infection also cause death to the S. exigua larva, where a total of 1.91 and 10.89% were infected by them, respectively. Whilst 16.64% of the collected larvae were dead due to pesticide and 7.44% were not known cause of the death. Besides that, 3.89% of the S. exigua died during pupal stage or emergence. Further, climatic factor was found not influencing the larva populations. There were no correlation between the number of larva collected with the means of temperature, relative humidity and rainfall.

Insecticide resistance poses many challenges in insect pest control, particularly in the control of destructive pests such as red imported fire ants (Solenopsis invicta). In recent years, beta-cypermethrin and fipronil have been extensively used to manage invasive ants, but their effects on resistance development in S. invicta are still unknown. To investigate resistance development, S. invicta was collected from populations in five different cities in Guangdong, China. The results showed 105.71- and 2.98-fold higher resistance against fipronil and beta-cypermethrin, respectively, in the Guangzhou population. The enzymatic activities of acetylcholinesterase, carboxylases, and glutathione S-transferases significantly increased with increasing beta-cypermethrin and fipronil concentrations. Transcriptomic analysis revealed 117 differentially expressed genes (DEGs) in the BC-ck vs. BC-30 treatments (39 upregulated and 78 downregulated), 109 DEGs in F-ck vs. F-30 (33 upregulated and 76 downregulated), and 499 DEGs in BC-30 vs. F-30 (312 upregulated and 187 downregulated). Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that DEGs associated with insecticide resistance were significantly enriched in metabolic pathways, the AMPK signaling pathway, the insulin signaling pathway, carbon metabolism, peroxisomes, fatty acid metabolism, drug metabolism enzymes and the metabolism of xenobiotics by cytochrome P450. Furthermore, we found that DEGs important for insecticide detoxification pathways were differentially regulated under both insecticide treatments in S. invicta. Comprehensive transcriptomic data confirmed that detoxification enzymes play a significant role in insecticide detoxification and resistance development in S. invicta in Guangdong Province. Numerous identified insecticide-related genes, GO terms, and KEGG pathways indicated the resistance of S. invicta workers to both insecticides. Importantly, this transcriptome profile variability serves as a starting point for future research on insecticide risk evaluation and the molecular mechanism of insecticide detoxification in invasive red imported fire ants.

In insects, cytochrome P450 monooxygenases (CYPs) play a pivotal role in the detoxification of various insecticides. The cigarette beetle, Lasioderma serricorne, is an economically important pest of stored products, for which pyrethroid insecticides are increasingly used. Nevertheless, the responses and functions of CYPs in L. serricorne under pyrethroid exposure remain poorly understood. In this study, three full-length CYP cDNAs were identified from L. serricorne and designated as CYP6BK29, CYP6SY1, and CYP6TE1. Phylogenetic analysis revealed that these CYPs belong to the CYP3 clan and the CYP6 family. Reverse transcription-quantitative PCR showed that CYP6BK29 and CYP6SY1 were predominantly expressed in the larval midgut, whereas CYP6TE1 transcripts were enriched in the fat body. The expression of CYP6BK29 peaked in the third- and fourth-instar larvae, while CYP6SY1 was specifically highest in the fourth-instar. In contrast, CYP6TE1 showed the highest mRNA levels in adults. Exposure to a sublethal dose (LD30) of lambda-cyhalothrin (LCT) significantly upregulated the mRNA levels of CYP6BK29 and CYP6TE1, but not that of CYP6SY1. RNAi-mediated knockdown of CYP6BK29 significantly increased the susceptibility of larvae to LCT. In contrast, silencing CYP6SY1 or CYP6TE1 had no significant effect on larval survival under LCT exposure. These results provide evidence that CYP6BK29 contributes to LCT detoxification in L. serricorne, highlighting its potential as a molecular target for novel pest management strategies.

Biosynthetic pathway of arachidonic acid in Spodoptera exigua in response to bacterial challenge

Spodoptera exigua nucleopolyhedrovirus (SeNPV) is an entomophathogenic virus of S. exigua larvae commonly used as bioinsecticide. This research was aimed to 1) study the symptoms of SeNPV infection on the S. exigua larvae in the laboratory, 2) examine the virulence of SeNPV on the 3rd instar of S. exigua larvae and (3) find out the optimal concentration of polyhedra and harvesting time. The infection of SeNPV on the S. exigua larvae was inhibited molting process and disturbing larval growth. The color of infected larvae gradually changed become more dark, and at the end of infection, larvae died with fragile and broken integument. Infected larvae showed reduction in feeding activities. The LC50 of SeNPV on 3rd instar larvae in the laboratory was estimated 6.65 x 105 POBs/ml. The polyhedra concentration used for virus propagation was 5.88 x 106 POBs/ml. The optimal harvesting time was 5 days after inoculation, where most of the infected larvae had died but the body still intact. Keywords: Pathology, symptom, virulence, Spodoptera exigua, SeNPV

Synthetic insecticides have been widely used for the prevention and control of disease vectors and agricultural pests. However, frequent uses of insecticides have resulted in the development of insecticide resistance in these insect pests. The resistance adversely affects the efficacy of insecticides, and seriously reduces the lifespan of insecticides. Therefore, resistance management requires new strategies to suppress insecticide resistance. Here, we confirm that CncC/Maf are the key regulators of various detoxification genes involved in insecticide resistance in Spodoptera exigua. Then, we develop a cell screening platform to identify the natural compound inhibitors of CncC/Maf and determine that sofalcone can act as a CncC/Maf inhibitor in vitro and in vivo. Bioassay results showed that sofalcone significantly enhanced the toxicity (more than 3-fold) of chlorpyrifos and lambda-cyhalothrin against S. exigua larvae. Finally, we demonstrate that sofalcone can greatly improve the susceptibility of S. exigua larvae to insecticides by inhibiting the activity of the ROS/CncC-dependent detoxifying enzymes and downregulating the expression levels of detoxification genes. CncC/Maf inhibitors can be used as broad-spectrum synergists to overcome insecticide resistance in pest populations. Altogether, our results demonstrate that reduced expression of detoxification genes resulting from suppression of transcriptional regulation of these genes contributes to controlling insecticide resistance, which provides a very novel and high-efficiency green resistance management strategy.

Spodoptera exigua (Hübner) is a polyphagous pest on agricultural crops, whose control is based mainly on the application of chemical insecticides. Bacillus thuringiensis (Bt) is one of the most important biological agents that have been successfully applied as a biological control, and Cry1Ca protein is considered to be active against S. exigua. Therefore, to understand the response of S. exigua to Cry1Ca protein, high-throughput sequencing was used to analyse the S. exigua larval midgut after treatment with sublethal concentrations of Cry1Ca protein. Transcriptome data showed that a total of 98,571 unigenes with an N50 value of 1135bp and a mean length of 653bp were obtained. Furthermore, 2962 differentially expressed genes (DEGs) were identified after Cry1Ca challenge, including 1508 up-regulated and 1454 down-regulated unigenes. Among these DEGs, detoxification (CYP, CarE, and GST) and Bt resistance (ALP, APN, and ABC transporter)-related genes were differentially expressed in the midgut of S. exigua after Cry1Ca treatment. However, most DEGs of protective enzymes were down-regulated, while most DEGs related with serine protease and REPAT were up-regulated. Furthermore, almost all DEGs related to the immune signaling pathway, antimicrobial protein, and lysozyme were up-regulated by Cry1Ca treatment. These results indicated that the detoxification enzyme, protective enzymes, Bt resistance-related genes, serine protease, REPAT, and the immune response might have been involved in the response of S. exigua to Cry1Ca protein. In summary, analysis of the transcriptomal expression of genes involved in Cry1Ca protein against S. exigua provided potential clues for elucidating the host response processes and defensive mechanisms underlying Cry1Ca toxicity.

This study examined the physiological effects of joint and separate parasitism and infection by the endoparasitoid Microplitis pallidipes Szépligeti and the nucleopolyhedrovirus (NPV), respectively, on haemolymph 20‐hydroxyecdysone (20‐E) titre in Spodoptera exigua (Hübner) larvae. The results indicated that in parasitized larvae, virus‐infected larvae (5.7 × 103 and 5.7 × 105 OB/ml) and parasitized larvae infected with virus at 5.7 × 105 OB/ml, compared to healthy larvae, the 20‐E all declined during the first 3 days but began to increase from day 4 after treatment, while in jointly parasitized and infected larvae (5.7 × 103 OB/ml), the 20‐E declined during the first 4 days but began to increase on day 5 after treatment. Meanwhile, compared to parasitized larvae, the 20‐E declined during the first 4 days but significantly increased on day 5 in jointly parasitized and infected larvae (5.7 × 103 OB/ml), while significantly increased during the first 2 days but began to decrease from day 3 after treatment in jointly parasitized and infected larvae (5.7 × 105 OB/ml). Finally, in larvae that were both parasitized and virus infected (5.7 × 103 OB/ml), compared to just virus‐infected larvae (5.7 × 103 OB/ml), the 20‐E was lower on days 3 and 4 but higher on other days after treatment; in larvae that were both parasitized and virus infected (5.7 × 105 OB/ml), compared to just virus‐infected larvae (5.7 × 105 OB/ml), the 20‐E was significantly higher at the first 2 days but lower from day 3 after treatment. Our results revealed that 2nd instar larval M. pallidipes in host bodies may release 20‐E into the haemolymph of S. exigua larvae and that NPV infection may stimulate S. exigua to release more 20‐E during its third to fourth instar larval moulting. We found that this stimulatory effect was greater with higher virus concentrations.

Why do Spodoptera exigua multiple nucleopolyhedrovirus occlusion bodies lose insecticidal activity on amaranth (Amaranthus hypocondriacus L.)?