几丁质合成酶 杀虫剂

Simple Summary In this study, we identified chitin synthase 1 gene (TcCHS1) from Tetranychus cinnabarinus (Boisduval) and then explored the gene expression levels of TcCHS1 at different developmental stages of T. cinnabarinus. We also investigated the effects of sublethal concentrations of diflubenzuron on the toxicities and survivals of T. cinnabarinus eggs and larvae as well as TcCHS1 expression levels. Our results demonstrated that TcCHS1 was essential for growth and development, and diflubenzuron exposure affected chitin metabolism. This work was undertaken to establish a foundation for further research on the functions of chitin synthase. It will provide a new target for controlling of T. cinnabarinus in the agricultural ecosystem. Abstract The carmine spider mite, Tetranychus cinnabarinus (Boisduval), is one of the most important acarine pest species. At present, its control remains primarily dependent on using various chemical insecticides/acaricides in agricultural crops worldwide. To clarify the mechanism whereby T. cinnabarinus responds to insecticide exposure, we identified the chitin synthase 1 gene (TcCHS1) and then explored the gene expression levels of TcCHS1 at different developmental stages of T. cinnabarinus. We also investigated the effects of sublethal concentrations of diflubenzuron on the toxicities and survivals of T. cinnabarinus eggs and larvae as well as TcCHS1 expression levels. The full-length cDNA sequence contains an open reading frame (ORF) of 4881 nucleotides that encoded for a 1474 amino acid residues protein. The predicted TcCHS1 protein had a molecular mass of 168.35 kDa and an isoelectric point of 6.26, and its amino acid sequence contained all the signature motifs (EDR, QRRRW and TWGTR) of chitin synthases. The results of phylogenetic analyses demonstrated that the putative CHS1 amino acid sequence of T. cinnabarinus revealed high similarities with chitin synthases in other insects and mites. Additionally, at the molecular level, transcriptional analysis by real-time quantitative PCR in different developmental stages of T. cinnabarinus revealed that TcCHS1 mRNA was expressed in all stages, and highest in eggs and female adults, but lowest in deutonymphs. Furthermore, the results of toxicity bioassays indicated that diflubenzuron treatment resulted in high mortality rates in eggs and larvae of T. cinnabarinus. The mRNA expression levels of TcCHS1 from the eggs and larvae of T. cinnabarinus were up-regulated in response to sublethal concentrations of diflubenzuron exposures. Together, all these results demonstrate that diflubenzuron has ovicidal and larvicidal effects and TcCHS1 may play an important role in the growth and development of T. cinnabarinus and may disrupt the chitin biosynthesis, thereby controlling T. cinnabarinus populations.

The brown planthopper (Nilaparvata lugens) is a major destructive rice pest in Asia. High levels of insecticide resistance have been frequently reported, and the G932C mutation in the chitin synthase 1 (CHS1) gene has been found to mediate buprofezin resistance. However, there has been no direct evidence to confirm the functional significance of the single G932C substitution mutation leading to buprofezin resistance in N. lugens. Here, we successfully constructed a knock-in homozygous strain (Nl-G932C) of N. lugens using CRISPR/Cas9 coupled with homology-directed repair (HDR). Compared with the background strain susceptible to buprofezin (Nl-SS), the knock-in strain (Nl-G932C) showed a 94.9-fold resistance to buprofezin. Furthermore, resistant strains (Nl-932C) isolated from the field exhibited a 2078.8-fold resistance to buprofezin, indicating that there are other mechanisms contributing to buprofezin resistance in the field. Inheritance analysis showed that the resistance trait is incomplete dominance. In addition, the Nl-G932C strain had a relative fitness of 0.33 with a substantially decreased survival rate, emergence rate, and fecundity. This study provided in vivo functional evidence for the causality of G932C substitution mutation of CHS1 with buprofezin resistance and valuable information for facilitating the development of resistance management strategies in N. lugens. This is the first example of using CRISPR/Cas9 gene-editing technology in a hemipteran insect to directly confirm the role of a candidate target site mutation in insecticide resistance.

The invasive species Aedes albopictus is a major vector of several arboviruses. The global spread of this species seriously threatens human health. Insecticide resistance is an increasing problem worldwide that limits the efficacy of mosquito control. As the major structural component of cuticles, chitin is indispensable to insects. Chitin synthase (CHS) is the enzyme that catalyzes the biosynthesis of chitin at the final step. In this study, two CHS genes of Aedes albopictus (AaCHS1 and AaCHS2) were identified and their basic characteristics were evaluated via bioinformatics analysis. The highest abundance of AaCHS1 transcripts was detected in pupae, whereas that of AaCHS2 transcripts was detected in females; the highest expression levels of AaCHS1 and AaCHS2 were found in the epidermis and the midgut of pupae, respectively. The survival and emergence rates of pupae were significantly reduced after the injection of double-stranded RNA of AaCHS1 or AaCHS2, indicating that both AaCHS1 and AaCHS2 play crucial roles in the pupal development. In addition, the chitin content of pupae was obviously decreased after the suppression of AaCHS1 expression by RNA interference (RNAi) treatment. This influence of the RNAi treatment was further supported by the reduced chitin thickness and weakened chitin fluorescence signal in the new cuticle. The midgut of pupae presented a reduced intensity of the chitin fluorescence signal along with RNAi treatment specific to AaCHS2 expression. The results of this study indicate that CHS genes may be suitable as molecular targets used for controlling mosquitoes.

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Simple Summary Lufenuron is one of the main insecticides for controlling lepidopteran pests, and is especially suitable for controlling pests that are resistant to pyrethroids and organophosphorus pesticides. Resistance to lufenuron is a serious obstacle to effective pest control. A major mechanism of lufenuron resistance is due to mutations in chitin synthase, the target of the toxic effects of benzoylurea (BPUs), according to a reported mutational analysis, I1040 (I1042 in Plutella xylostella) in the SfCHSA gene. The mutation of the site can significantly improve the resistance of insect larvae to BPU insecticides. Secondly, the expression level of insecticide targets in insects is also an important factor affecting insect resistance. In this study, we firstly identified the number of chitin synthase genes and their corresponding names in Spodoptera frugiperda, and detected the mutation sites of the SfCHSA gene. The expression level of chitin synthase gene was then analyzed. Our results showed that no mutation was found in the SfCHSA gene in the currently low-resistant field populations in China, and the expression of chitin synthase was affected by lufenuron. The information obtained from this study is valuable for the use of lufenuron in the field. Abstract Spodoptera frugiperda (J. E. Smith), is commonly known as fall armyworm, native to tropical and subtropical regions of America, is an important migratory agricultural pest. It is important to understand the resistance and internal mechanism of action of S. frugiperda against lufenuron in China. Lufenuron is one of the main insecticides recommended for field use in China and has a broad prospect in the future. We conducted a bioassay using the diet-overlay method and found that the current S. frugiperda in China are still at a low level of resistance to lufenuron. Secondly, we examined whether the mutation I1040M (I1042M in Plutella xylostella), associated with lufenuron resistance, was produced in the field. And then we tested the expression of chitin synthase SfCHSA and SfCHSB in different tissues, and the changes of these two genes after lufenuron induction. The results showed that there is still no mutation generation in China and there is a significant change in the expression of SfCHSA under the effect of lufenuron. In conclusion, our study suggests that field S. frugiperda populations in 2019 and 2020 were less resistant to lufenuron. In fall armyworm, chitin synthases included SfCHSA and SfCHSB genes, and after induction treatment with lufenuron, the expression of the SfCHSA gene was significantly increased. In SfCHSA, no mutation has been detected in the site associated with lufenuron resistance. Secondly, in S. frugiperda larvae, the SfCHSA gene was the highest in the head of the larvae, followed by the integument; while the SfCHSB gene was mainly concentrated in the midgut. Therefore, we believe that the SfCHSA gene plays a greater role in the resistance of S. frugiperda to lufenuron than the SfCHSB gene. It is worth noting that understanding the level of resistance to lufenuron in China, the main mechanism of action of lufenuron on larvae, and the mechanism of resistance to lufenuron in S. frugiperda will help in crop protection as well as in extending the life span of this insecticide.

The pathogens transmitted by mosquitoes to humans and animals cause several emerging and resurgent infectious diseases. Increasing insecticide resistance requires rational action to control the target vector population. Chitin is indispensable for insect growth and development and absent from vertebrates and higher plants. Chitin synthase A (CHSA) is a crucial enzyme in chitin synthesis; therefore, identifying and characterizing how CHSA determines chitin content may contribute to the development of novel vector control strategies. The injection of small interfering RNA targeting CHSA (siCHSA) to knockdown CHSA transcripts in larval, pupal and adult stages of Culex pipiens pallens resulted in the appearance of different lethal phenotypes. When larval and pupal stages were injected with siCHSA, CHSA knockdown prevented larval molting, pupation and adult eclosion, and affected the production of chitin and chitin degradation, which resulted in an ecdysis defect phenotype of mosquitoes. When siCHSA was injected into mosquitoes in the adult stage, CHSA knockdown also affected the laminar organization of the mesoderm and the formation of pseudo-orthogonal patterns of the large fibers of the endoderm. We provide a systematic and comprehensive description of the effects of CHSA on morphogenesis and metamorphosis. The results show that CHSA not only affects chitin synthesis during molting, but also might be involved in chitin degradation. Our results further show that CHSA is important for the structural integrity of the adult mosquito cuticle.

Background: The pathogens transmitted by mosquitoes (Culex pipiens pallens) to humans and animals cause several emerging and resurgent infectious diseases. Increasing insecticide resistance requires rational action to control the target vector population. Chitin is indispensable for insect growth and development and absent from vertebrates and higher plants. Chitin synthase A (CHSA) represents a crucial enzyme in chitin synthesis; therefore, identifying and characterizing how CHSA determines the chitin content might help with novel vector control strategies. Results: The injection of small interfering RNA targeting CHSA (siCHSA) to knock down CHSA transcripts of in larval, pupal, and adult stages, showed different lethal phenotypes. In the larval and pupal stages, CHSA knockdown prevented larval molting, pupation, and adult eclosion, and affected the production of chitin and chitin degradation, which resulted in an ecdysis defect phenotype of mosquitoes. In the adult stage, it also affected the laminar organization of mesoderm and the formation of pseudo orthogonally large fibers of the endoderm. Conclusion: The present study provides a systematic and comprehensive description of the effects of CHSA on morphogenesis and metamorphosis. The results showed that CHSA not only affects chitin synthesis during molting, but also might be involved in chitin degradation. Our result further showed that CHSA is important for the structural integrity of the adult mosquito cuticle.

Background Diflubenzuron (DFB) is one of the most used insecticides in mosquito larval control including that of Culex pipiens, the proven vector of the recent West Nile Virus epidemics in Europe. Two mutations (I1043L and I1043M) in the chitin synthase (CHS) putative binding site of DFB have been previously reported in Cx. pipiens from Italy and associated with high levels of resistance against this larvicide. Methodology/Principal findings Here we report the identification of a third mutation at the same I1043 position of the CHS gene resulting in the substitution of Isoleucine to Phenylalanine (I1043F). This mutation has also been found in agricultural pests and has been functionally validated with genome editing in Drosophila, showing to confer striking levels (>15,000 fold) of DFB resistance. The frequency of the I1043F mutation was found to be substantially higher in Cx. pipiens mosquitoes surviving DFB doses largely exceeding the recommended field dose, raising concerns about the future efficient use of this insecticide. We monitored the presence and frequency of DFB mutations in Cx. pipiens mosquitoes from several Mediterranean countries, including Italy, France, Greece, Portugal and Israel. Among the Cx. pipiens populations collected in Northern Italy all but one had at least one of the three DFB mutations at allele frequencies reaching 93.3% for the I1043M, 64.8% for the I1043L and 10% for the I1043F. The newly reported I1043F mutation was also identified in two heterozygote individuals from France (4.2% allelic frequency). In contrast to Italy and France, no DFB resistant mutations were identified in the Cx. pipiens mosquitoes sampled from Greece, Portugal and Israel. Conclusions/Significance The findings of our study are of major concern for mosquito control programs in Europe, that rely on the use of a limited number of available larvicides, and highlight the necessity for the development of appropriate Insecticide Resistance Management (IRM) programs, to ensure the sustainable use of DFB.

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Insecticide resistance is one of the primary problems affecting vector control worldwide. Assessing the occurrence of resistant alleles and understanding their origin across the geographic range of vector species is crucial for effective resistance management. In populations of the mosquito Culex pipiens, point mutations conferring resistance to the insecticide diflubenzuron (DFB) were recently found across the Mediterranean basin. In this study, we investigated the possible occurrence of DFB resistance in Cyprus, where West Nile virus outbreaks have been documented in recent years. We sequenced a fragment of the chitin-synthase 1 gene carrying the resistant mutations in individuals collected from 18 populations of Cx. pipiens in Cyprus to investigate the occurrence of DFB-resistant alleles. We then assessed the evolutionary origin of DFB-resistant alleles by reconstructing the phylogenetic relationships between susceptible and resistant alleles found across the Mediterranean basin. Our screening revealed the occurrence of the I1043F allele in all the districts analyzed. Notably, a new gene codon underlying the I1043F allele was detected. To our knowledge, this has not been previously reported in areas with DFB-resistance alleles in Cx. pipiens. In addition, we observed that the I1043F alleles detected in Cyprus have a different genetic background from those reported in other geographic areas, such as Italy and Turkey. To our knowledge, this is the first time in which DFB resistance was revealed in Cx. pipiens populations occurring in Cyprus. Furthermore, we demonstrate that I1043F-resistant alleles have an independent origin in Cyprus, further supporting the hypothesis of a multiple independent origin of DFB resistance across the Mediterranean region. These results stress the need for regular resistance surveillance activities and the urgency of developing new mosquito control strategies.

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BACKGROUND Previous studies have shown that fungicides have insecticidal activity that can potentially be used as an insecticide resistance management strategy in the brown planthopper Nilaparvata lugens (Stål). However, the mechanism that induces mortality of N. lugens remains elusive. RESULTS In the present study, the insecticidal activities of 14 fungicides against N. lugens were determined, of which tebuconazole had the highest insecticidal activity compared with the other fungicides. Furthermore, tebuconazole significantly inhibited the expression of the chitin synthase gene NlCHS1; the chitinase genes NlCht1, NlCht5, NlCht7, NlCht9, and NlCht10; and the β-N-acetylhexosaminidase genes NlHex3, NlHex4, NlHex5 and NlHex6; it significantly suppressed the expression of ecdysteroid biosynthetic genes as well, including SDR, CYP307A2, CYP307B1, CYP306A2, CYP302A1, CYP315A1 and CYP314A1 of N. lugens. Additionally, tebuconazole affected the diversity, structure, composition, and function of the symbiotic fungi of N. lugens, as well as the relative abundance of saprophytes and pathogens, suggesting that tebuconazole reshapes the diversity and function of symbiotic fungi of N. lugens. CONCLUSION Our findings illustrate the insecticidal mechanism of tebuconazole, possibly by inhibiting normal molting or disrupting microbial homeostasis in N. lugens, and provide an important rationale for developing novel insect management strategies to delay escalating insecticide resistance. This article is protected by copyright. All rights reserved.

Background Insect growth regulators (IGRs) are considered a novel group of insecticides to control mosquitoes. Novaluron is an IGR with benzoylphenyl urea insecticide, which inhibits chitin synthesis in insects and can reduce insect population density; it is also known to have a high margin of safety for mammals. Methods The effective minimum concentration of novaluron formulation EC10 was tested. Six pineapple plantations [control ( n  = 3) and test ( n  = 3)] were selected from Meerigama Medical Officer of Health area in Gampaha District, Sri Lanka. Fifteen plots (10 × 10 m) were demarcated in each site with a 200 m distance apart. Leaf axils of 450 pineapple plants (30 plants × 15 plots) were screened for immature stages of Aedes mosquitoes weekly for 12 weeks. The required concentration (20 ppm) of novaluron was sprayed onto the selected pineapple plants ( n  = 1350) individually in 3 selected test sites for 5–10 s. The reduction in the vector population was interpreted as the percentage of reduction in immature stages of Aedes mosquitoes. Results The 100% mortality of the Ae. aegypti larvae within 24 h was observed at 20 ppm (0.05 ml of novaluron 100 g/l in 250 ml of water) as the minimum dose. Variation in the number of Aedes larvae present in the control and intervention sites was found to be significantly different throughout the entire observational period ( χ 2  = 128.29, df  = 11, P  < 0.001). The total elimination of Aedes larvae continued for up to 2 weeks and a 50% reduction was observed until the 8th week. Conclusions The present study emphasizes that novaluron (10% EC) can be used as an effective larvicide at the treatment dose of 20 ppm. The residual effect of the IGR lasted for 12 weeks with a functional efficacy of 8 weeks. The 100% reduction of larval breeding was observed up to the 2nd week after application and the percentage reduction of immature stages remained > 50% until the 8th week. The lowest reduction (34.2%) was observed at 12 weeks after the initial treatment. Therefore, re-treatment may be recommended based on the reduction in the efficacy of the IGR.

Chemical control of dengue vector, Aedes aegypti is impaired due to development of resistance to conventional insecticides. Insect Growth Regulators (IGRs) are considered more suitable and effective vector control agents as they specifically inhibit chitin biosynthesis, a process absent in vertebrates, and impose less adverse effects on beneficial insects and the environment. Present study investigates Lufenuron, a Chitin Synthesis Inhibitor (CSI), as a control agent of Ae. aegypti. Different instars of Ae. aegypti were exposed to a range of concentrations of Lufenuron as per WHO protocol. The investigations showed the effective hormone-mimetic effect of Lufenuron resulting in the formation of a significant number of larval-pupal and pupal-adult intermediates with the maximum number observed on exposure to L3 (L-P=17%, P-A=21%). Approximately 20% of L2 instars either could not moult and remained trapped inside the new exuviae or possessed bulged abdomen while some showed ruptured exoskeleton. The results showed increase in IE30 from L1 (0.00010 ppm) to L4 stage (0.00013 ppm); the L2 stage exhibiting maximum IE30 (0.00025 ppm). The median emergence suppression (IE50) doses of the Lufenuron were found to be 0.00057 ppm for L1, 0.00047 ppm for L2, 0.00050 ppm for L3 and 0.00096 ppm for L4. The results also revealed increased duration of larval development and inability of pupae to develop into adults, as compared to the controls. The investigations indicate the potential use of Lufenuron as the control agent of Ae. aegypti. Further research is being conducted to understand its mode of action to develop effective control strategies.

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Lufenuron, a benzoylurea chitin synthesis inhibitor, is effective against many insect pests. However, the insecticidal activity of lufenuron has not been completely elucidated, nor has its disturbing effect on chitin synthesis genes. In this study, bioassay results demonstrated an outstanding toxicity of lufenuron against Helicoverpa armigera larvae. The treated larvae died from abortive molting and metamorphosis defects, and severe separation of epidermis and subcutaneous tissues was observed. Treatment of 3rd- and 4th-instar larvae with LC25 lufenuron significantly extended the duration of larval and pupal stage, reduced the rates of pupation and emergence, and adversely affected pupal weight. Besides, lufenuron can severely reduce chitin content in larval integument, and the lufenuron-treated larvae showed reduced trehalose content in their hemolymph. Further analysis using RNA sequencing revealed that five chitin synthesis genes were down-regulated, whereas the expressions of two chitin degradation genes were significantly enhanced. Knockdown of chitin synthase 1 (HaCHS1), uridine diphosphate-N-acetylglucosamine-pyrophosphorylase (HaUAP), phosphoacetyl glucosamine mutase (HaPGM), and glucosamine 6-phosphate N-acetyl-transferase (HaGNPAT) in H. armigera led to significant increase in larval susceptibilities to LC25 lufenuron by 75.48%, 65.00%, 68.42% and 28.00%, respectively. Our findings therefore revealed the adverse effects of sublethal doses of lufenuron on the development of H. armigera larvae, elucidated the perturbations on chitin metabolism, and proved that the combination of RNAi and lufenuron would improve the control effect of this pest.

Наличие кишечного, контактного, овицидного, ларвицидного, стерилизующего и морфогенетического действия у соединений из группы регуляторов развития насекомых (РРН) позволяет использовать их так же широко, как и инсектициды [1–3; 9]. Отсутствие у РРН острого действия, характерного для традиционных инсектицидов, ограничивает их широкое применение. Если за рубежом в качестве ларвицидов преимущественно используют РРН как более безопасные соединения для объектов окружающей среды, то у нас в стране только недавно появились единичные производители, которые начали выпускать препараты этого типа. Оригинальность механизма действия, широкий спектр активности, длительность (до нескольких месяцев) остаточного эффекта позволяют использовать их как самостоятельно, так и в сочетании традиционными инсектицидами в многокомпонентных системах борьбы. The presence of intestinal, contact, ovicidal, larvicidal, sterilizing and morphogenetic effects of a compound from the group of insect growth regulators (IGR) allows their use as widely as insecticides [1-3; 9]. The lack of acute action of PPH, inherent in traditional insecticides limits their widespread use. If abroad, IGR is preferably used as safer compounds for environmental objects as larvicides, in our country there are only a few manufacturers who have begun to produce drugs of this type. The originality of the mechanism of action, a wide spectrum of activity, the duration (up to several months) of the residual effect allows using them both independently and in combination with traditional insecticides in multi-component control systems.

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BackgroundResistance to traditional insecticides represents a threat to the control of disease vectors. The insect growth regulators (IGR) are a potential alternative to control mosquitoes, including resistant populations. The chitin synthesis inhibitors (CSI) are IGRs, which interfere with the insect molting process and represent one major class of compounds against Aedes aegypti populations resistant to the larvicide organophosphate temephos. In the present study, we evaluated the efficacy of the CSI triflumuron on Culex quinquefasciatus, Aedes albopictus and against several Ae. aegypti field populations.MethodsThe efficacy of triflumuron, against Cx. quinquefasciatus and Ae. albopictus was evaluated with laboratory strains through dose–response assays. Additionaly, this CSI was tested against seven Ae. aegypti field populations exhibiting distinct resistance levels to both temephos and the pyrethroid deltamethrin. Aedes aegypti populations were exposed to both a dose that inhibits 99% of the adult emergence of mosquitoes from the susceptible reference strain, Rockefeller, (EI99 = 3.95 μg/L) and the diagnostic dose (DD), corresponding to twice the EI99.ResultsOur results indicate that triflumuron was effective in emergence inhibition (EI) of Cx. quinquefasciatus (EI50= 5.28 μg/L; EI90= 12.47 μg/L) and Ae. albopictus (EI50= 1.59 μg/L; EI90= 2.63 μg/L). Triflumuron was also effective against seven Ae. aegypti Brazilian populations resistant to both temephos and deltamethrin. Exposure of all the Ae. aegypti populations to the triflumuron EI99 of the susceptible reference strain, Rockefeller, resulted in complete inhibition of adult emergence, suggesting no cross-resistance among traditional insecticides and this CSI. However, a positive correlation between temephos resistance and tolerance to triflumuron was observed.ConclusionThe results suggest that triflumuron represents a potential tool for the control of disease vectors in public health. Nevertheless, they point to the need of constant monitoring of the susceptibility status of vector populations to CSIs.

Population control of the dengue vector mosquito, Aedes aegypti, is difficult due to many reasons, one being the development of resistance to neurotoxic insecticides employed. The biosynthesis of chitin, a major constituent of insect cuticle, is a novel target for population control. Novaluron is a benzoylphenylurea (BPU) that acts as a chitin synthesis inhibitor, already used against mosquitoes. However, information regarding BPU effects on immature mosquito stages and physiological parameters related with mosquito larval development are scarce. A set of physiological parameters were recorded in control developing larvae and novaluron was administered continuously to Ae. aegypti larvae, since early third instar. Larval instar period duration was recorded from third instar until pupation. Chitin content was measured during third and fourth instars. Fourth instars were processed histochemically at the mesothorax region, stained with hematoxylin and eosin (HE) for assessment of internal tissues, and labeled with WGA-FITC to reveal chitinized structures. In control larvae: i) there is a chitin content increase during both third and fourth instars where late third instars contain more chitin than early fourth instars; ii) thoracic organs and a continuous cuticle, closely associated with the underlying epidermis were observed; iii) chitin was continuously present throughout integument cuticle. Novaluron treatment inhibited adult emergence, induced immature mortality, altered adult sex ratio and caused delay in larval development. Moreover, novaluron: i) significantly affected chitin content during larval development; ii) induced a discontinuous and altered cuticle in some regions while epidermis was often thinner or missing; iii) rendered chitin cuticle presence discontinuous and less evident. In both control and novaluron larvae, chitin was present in the peritrophic matrix. This study showed quantitatively and qualitatively evidences of novaluron effects on Ae. aegypti larval development. To our knowledge, this is the first report describing histological alterations produced by a BPU in immature vector mosquitoes.

Abstract The effects of sublethal concentrations 0.1, 0.5, and 1.2 µg mL-1of the chitin synthesis inhibitor, hexaflumuron, on larval growth and development, the count and proportion of hemocytes, and carbohydrate content (trehalose and glyceride) in hemolymph were investigated in the cutworm, Spodoptera litura (Fabricious) (Lepidoptera: Noctuidae). When 3rdinstar larvae were subjected to the sublethal concentrations, there were dose-dependent effects on larval weight and length of each instar larvae, percent pupation and the duration of development. Most of the larvae died during the molting process at all concentrations. Few individuals from 0.5 and 1.2 µg mL -1concentrations could develop to the 6thinstar, while the pupae emerging from the 0.1 µg mL -1concentrations did not exceed 16% of the number of the initial larvae. In 5thinstar S. litura, the total number of hemocytes was significantly increased at 24 hours post—treatment, whereas the proliferation of hemocytes was inhibited, plasmatocyte pseudopodia contracted, and granulocyte expanded at 96 hours post—treatment. The increases of plasmatocyte count and the decreases of granulocyte count were dose—dependent. The longer treatment time of the sublethal concentrations increased the content of total carbohydrate and trehalose in hematoplasma, and was dose—dependent in hemocytes. The content of glyceride in hemolymph was significantly higher at 24 hours post—treatment, but gradually returned to normal levels at 96 hours post—treatment as compared with the control. The results suggested that sublethal concentrations of hexaflumuron reduced S. litura larval survival and interfered with hemolymph physiological balances.

The vegetative insecticidal protein Vip3Aa from Bacillus thuringiensis (Bt) has been produced by transgenic crops to counter pest resistance to the widely used crystalline (Cry) insecticidal proteins from Bt. To proactively manage pest resistance, there is an urgent need to better understand the genetic basis of resistance to Vip3Aa, which has been largely unknown. We discovered that retrotransposon-mediated alternative splicing of a midgut-specific chitin synthase gene was associated with 5,560-fold resistance to Vip3Aa in a laboratory-selected strain of the fall armyworm, a globally important crop pest. The same mutation in this gene was also detected in a field population. Knockout of this gene via CRISPR/Cas9 caused high levels of resistance to Vip3Aa in fall armyworm and 2 other lepidopteran pests. The insights provided by these results could help to advance monitoring and management of pest resistance to Vip3Aa.

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The chitin synthase gene 1 (CHS1) is a key gene in insect chitin synthesis pathway, it plays a critical role in the insect's survival and development. However, the protective functions of CHS1 in response to pathogens and chemical insecticides remain poorly understood. In this study, we analyzed the functional domain and phylogenetic relationship of CHS1 in Nilaparvata lugens and other insects. Our findings revealed a conserved C-terminal domain in the CHS1 protein, as well as an evolutionary conservation across insect species. And then we found the CHS1 gene was highly expressed during the fifth instar nymph stage, and there was a differential expression and regulation of CHS1 in response to pathogen infection and exposure to various chemical insecticides. After that, we further discovered RNA interference (RNAi) mediated knockdown of CHS1 significantly increased the susceptibility of N. lugens to Cordyceps javanica and two chemical insecticides, nitenpyram and dinotefuran, but had no effect on triflumezopyrim. And we used scanning electron microscope to observe an increase in appressoria formation on the cuticle of N. lugens following CHS1 knock down, which accelerated the infection by C. javanica. These findings showed that CHS1 in N. lugens provide protection against pathogen and chemical insecticides, and highlighted the potential of targeting CHS1 to develop novel pest management strategies.

Genetically engineered crops that produce insecticidal proteins from Bacillus thuringiensis (Bt) have many benefits and are used globally to manage key insect pests, including Helicoverpa zea (Lepidoptera: Noctuidae), a major pest of crops in the Americas. However, pests of at least 11 species, including H. zea, have evolved resistance to Bt crops, diminishing their effectiveness and benefits. For H. zea in the United States, practical resistance to Bt corn and cotton producing crystalline (Cry) Bt proteins is widespread and early warning of resistance to the vegetative insecticidal protein Vip3Aa has been reported. Thus, a better understanding of the genetic basis of resistance to Vip3Aa is needed to monitor, manage and counter resistance. In some strains of lepidopteran pests, resistance to Vip3Aa is associated with disruptive mutations in the chitin synthase 2 (CHS2) gene but this association had not been investigated previously in H. zea.

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Simple Summary Insect pests are a major problem worldwide and their control represents an urgency for human well-being. Insecticides acting as chitin-synthesis inhibitors (CSIs) are one of the major tools used to control these insects, but resistance is evolving in many species against several chemical classes, such as oxazolines and benzoylureas. Genetic studies showed that resistance is associated with point mutations in position 1043 of the chitin-synthase 1 gene (chs1), which change an isoleucine into phenylalanine, leucine or methionine (i.e., I1043F; I1043L; I1043M, respectively). Here, we studied the molecular and phenotypic characteristics of the homozygous I1043M strain in the mosquito Culex pipiens by comparing susceptible and resistant individuals. Our results showed that resistant mosquitoes have a striking level of resistance when exposed to the CSI diflubenzuron. Furthermore, cuticle modifications and the over-expression of the chs1 gene were detected in resistant Cx. pipiens, which are associated and, thus, likely contribute to resistance, as well as to the intensity of the resistant phenotype. Since mutations on the chs1 gene are conserved among different arthropod pest species, our results could be valuable to understand CSI resistance not only in mosquitoes, but also in a wider perspective. Abstract Insecticide resistance is a major threat challenging the control of harmful insect species. The study of resistant phenotypes is, therefore, pivotal to understand molecular mechanisms underpinning insecticide resistance and plan effective control and resistance management strategies. Here, we further analysed the diflubenzuron (DFB)-resistant phenotype due to the point-mutation I1043M in the chitin-synthase 1 gene (chs1) in the mosquito Culex pipiens. By comparing susceptible and resistant strains of Cx. pipiens through DFB bioassays, molecular analyses and scanning electron microscopy, we showed that the I1043M-resistant mosquitoes have: (i) a striking level of DFB resistance (i.e., resistance ratio: 9006); (ii) a constitutive 11-fold over-expression of the chs1 gene; (iii) enhanced cuticle thickness and cuticular chitin content. Culex pipiens is one of the most important vector species in Europe and the rapid spread of DFB resistance can threaten its control. Our results, by adding new data about the DFB-resistant phenotype, provide important information for the control and management of insecticide resistance.

The pea aphid, Acyrthosiphon pisum, is an important agricultural pest and an ideal model organism for various studies. Chitin synthase (CHS) catalyses chitin synthesis, a critical structural component of insect exoskeletons. Here, we identified a CHS gene from A. pisum, ApisCHS. The ApisCHS expression profiles showed that ApisCHS was expressed in various developmental stages and in all tested tissues of A. pisum, including the epidermis, embryo, gut and haemolymph. Notably, ApisCHS exhibited peak expression in the middle of each nymphal period and was extremely highly expressed in the epidermis and embryo. RNA interference (RNAi) showed that ~600 ng of dsRNA is an effective dose for gene silencing by injection for dsRNA delivery; moreover, 1200 ng·μL−1 dsRNA induced CHS gene silencing by a plant-mediated feeding approach. A 44.7% mortality rate and a 51.3% moulting rate were observed 72 h after injection of dsApisCHS into fourth-instar nymphs, compared with the levels in the control (injected with dsGFP). Moreover, a longer period was required for nymph development and a 44.2% deformity rate among newborn nymphs was obtained upon ingestion of dsApisCHS. These results suggest that ApisCHS plays a critical role in nymphal growth and embryonic development in pea aphids, and is a potential target for RNAi-based aphid pest control.

Simple Summary Heortia vitessoides Moore is a leaf-eating pest that affects Aquilaria sinensis. In the outbreak period, the leaves of Aquilaria sinensis can be eaten up in a short time, resulting in the death of trees and great economic losses. Chitin is the main component of insect cuticle, peritrophic membrane, and tracheal intima. Chitin synthesis in insects is a complex process that requires the cooperation of many enzymes. Chitin synthase is one of the key enzymes in the process. Chitin synthase is divided into two types: chitin synthase A gene (Chsa) and chitin synthase B gene (Chsb). Chsb is mainly responsible for the tissue synthesis of chitin in midgut peritrophic membrane. It was found that the expression of HvChsb was inhibited, the growth and development were abnormal, and the mortality rate was increased. These findings provide a reference for the prevention and control of the pest from the perspective of gene manipulation. Abstract The chitin synthase B gene is a key enzyme in the chitin synthesis of insect peritrophic matrix (PM), which affects insects’ feeding behavior. The chitin synthase B gene was cloned from the transcription library of Heortia vitessoides Moore. RT-qPCR showed that HvChsb was highly expressed in the larval stage of H. vitessoides, especially on the first day of the pre-pupal stage, as well as in the midgut of larvae and the abdomen of adults. After starvation treatment, HvChsb was found to be significantly inhibited over time. After 48 h of starvation, the feeding experiment showed that HvChsb increased with the prolongation of the re-feeding time. The experimental data showed that feeding affected the expression of HvChsb. HvChsb was effectively silenced via RNA interference; thus, its function was lost, significantly decreasing the survival rate of H. vitessoides. The survival rate from larval-to-pupal stages was only 43.33%, and this rate was accompanied by abnormal phenotypes. It can be seen that HvChsb plays a key role in the average growth and development of H. vitessoides.

The peritrophic matrix (PM) serves as a crucial intestinal barrier in insects, with Chitin Synthase 2 (CHS2) and Insect Intestinal Mucin (IIM) being its essential structural components. However, the regulatory mechanisms controlling their expression remain poorly understood. Here, we identified HaIIM80 as the most highly expressed chitin-binding protein gene in the midgut of Helicoverpa armigera larvae. Through dual-luciferase assays, DNA pull-down, RNA interference, histological assays, and functional studies, we demonstrated that the transcription factor HaGATAe binds to promoters of both HaCHS2 and HaIIM80 via multiple GATA sites, upregulating their expression and promoting PM integrity. Knockdown of HaGATAe reduced midgut chitin abundance and PM thickness, increased susceptibility to HaNPV, but decreased sensitivity to Vip3Aa toxin. Furthermore, the ecdysone-responsive factors HaEcRb enhanced the HaGATAe-mediated activation of HaCHS2 and HaIIM80, and HaHR3 suppressed them, which is consistent with their expression patterns during pupation period. This regulatory mode was further supported by RNAi of HR3, starvation, and treatment with the ecdysone agonist methoxyfenozide, collectively indicating that ecdysone signaling regulates the expression of both HaCHS2 and HaIIM80. Besides, HaE75C (an HR3 repressor) and its ligand heme upregulated the expression of both HaCHS2 and HaIIM80. These findings reveal a transcriptional regulatory network affecting PM formation and provide potential targets for pest management strategies aiming at disrupting gut barrier function.

Elaborate regulation of tissue- and stage-specific expression of genes is prerequisite for insect development. The hormone 20-hydroxyecdysone (20E) initiates metamorphosis by regulating the expression of a series of genes. However, how 20E orderly regulates the pupa-specific expression of genes remains unclear. In this study, we report a regulatory mechanism for the pupa-specific expression of chitin synthase A 2b (CHSA-2b) in Bombyx mori. We found that Broad-Complex Z4 (BR-C Z4) was up-regulated by 20E just before pupation, while transcription factor FoxJ and CHSA-2b were up-regulated during the pupal stage. There is a Fox cis-regulatory element in the CHSA-2b promoter region, and FoxJ protein bound to this element, enhancing the CHSA-2b transcription during the pupal stage. In addition to CHSA-2b, FoxJ also up-regulated the expression of 16 out of 19 pupa-specific genes tested. However, at the prepupal stage, 20E-induced BR-C Z4 inhibited the FoxJ transcription, indirectly inhibiting the CHSA-2b transcription. These data suggest that at the pre-pupation stage, 20E-induced BR-C Z4 inhibited the expression of pupa-stage genes like CHSA-2b by inhibiting the expression of FoxJ; by the pupal stage, the expression of BR-C Z4 decreased, releasing its inhibition on FoxJ, which then up-regulated the expression of the pupa-specific genes. This study explains the elaborate regulation of the pupa-specific gene expression during metamorphosis in B. mori.

Brown planthopper (Nilaparvata lugens) is one of the important pests that damage rice. Trehalose‐6‐phosphate synthase (TPS) is a key enzyme responsible for catalysing the biosynthesis of trehalose, which is the energy substance of insects. In this study, combined with the reported N. lugens TPS1, TPS2 and newly discovered TPS3, we studied the regulation of TPS in chitin metabolism by RNA interference. Firstly, we found that the relative expression levels of TRE1‐1, TRE1‐2 and TRE2 increased significantly after 48 h of dsTPS3 injection, and the activity of TRE1 enhanced significantly. Secondly, abnormal and lethal phenotypes were observed after dsTPS3 and dsTPSs injection. The relative expression levels of PGM2, G6PI2, Cht1‐4, Cht6‐10 and IDGF decreased significantly after 48 h of dsTPS3 injection. At 72 h after injection of dsTPS3, the relative expression levels of CHS1, Cht2, Cht4, Cht7 and Cht8 reduced significantly, but the expression levels of G6PI1, Cht5 and ENGase increased significantly. The relative expression levels of GFAT, UAP, PGM2, G6PI2, CHS1, CHS1a, CHS1b, Cht2, Cht4, Cht8, Cht9 and Cht10 decreased significantly after 48 h of dsTPSs injection. However, at 72 h after the injection of dsTPSs, the expression levels of GNPNA, UAP, PGM1, G6PI1, HK, CHS1, CHS1a, CHS1b, Cht3, Cht5, Cht7 and ENGase increased significantly. Finally, the chitin content decreased in dsTPS1, dsTPS2 and dsTPSs treatments. In conclusion, the inhibition of TPS expression affected the metabolism of trehalose and chitin in N. lugens. The related research results provide a theoretical basis for pest control.

Simple Summary Liriomyza trifolii is an important insect pest that infects many horticultural crops and vegetables, displaying strong interspecific competitiveness and inflicting serious harm. Here, chitin synthase 2 transcript was studied, and a prepupal immersion dsRNA delivery method was established for L. trifolii. The dsRNA can enter in the prepupal stage and play a role in the pupal stage, resulting in a decrease in the CHS2 expression level and eclosion rate. This study is an important supplement to the research on the RNAi of L. trifolii, enhances knowledge of the function of chitin synthase 2 in Liriomyza species, and may also provide a new idea for its control strategies. Abstract Liriomyza trifolii is an important invasive pest that infects horticultural vegetables, displaying a strong competitive advantage and showing great potential for inflicting harm. Chitin synthase is one of the key enzymes in insect chitin metabolism and plays an important role in insect growth and development. In this study, a chitin synthase (CHS) transcript of L. trifolii was cloned, and the results showed that LtCHS belongs to the CHS2 family. The expression analysis indicated the presence of the highest abundance of LtCHS2 in the pupae at different developmental stages but showed no significant difference among different tissues in the adult. Furthermore, a dsRNA immersion method was developed for RNA interference (RNAi) in L. trifolii using LtCHS2 transcript. RNAi can significantly reduce the expression of LtCHS2 in pupae, and the emergence rate of the pupae was significantly lower than that of the control. The results provide a theoretical basis for exploring the role of chitin synthase gene in L. trifolii and proposing new pest control strategies.

No abstract available

Chitin is a vital part of the insect exoskeleton and peritrophic membrane, synthesized by chitin synthase (CHS) enzymes. Chitin synthase 1 (CHS1) is a crucial enzyme in the final step of chitin biosynthetic pathway and consequently plays essential role towards insect growth and molting. RNA interference (RNAi) is an agent that could be used as an extremely target-specific and ecologically innocuous tactic to control different insect pests associated with economically important crops. The sole purpose of the current study is to use CHS1 as the key target gene against the cotton-melon aphid, Aphis gossypii, via oral feeding on artificial diets mixed with dsRNA-CHS1. Results revealed that the expression level of CHS1 gene significantly decreased after the oral delivery of dsRNA-CHS1. The knockdown of CHS1 gene caused up to 43%, 47%, and 59% mortality in third-instar nymph after feeding of dsCHS1 for 24, 48, and 72 h, respectively, as compared to the control. Consistent with this, significantly lower longevity (approximately 38%) and fecundity (approximately 48%) were also found in adult stage of cotton-melon aphids that were fed with dsCHS1 for 72 h at nymphal stage. The qRT-PCR analysis of gene expression demonstrated that the increased mortality rates and lowered longevity and fecundity of A. gossypii were attributed to the downregulation of CHS1 gene via oral-delivery-mediated RNAi. The results of current study confirm that CHS1 could be an appropriate candidate target gene for the RNAi-based control of cotton-melon aphids.

Trehalose‐6‐phosphate synthase (TPS), an enzyme that hydrolyzes two glucose molecules to yield trehalose, plays a pivotal role in various physiological processes. In this study, we cloned the trehalose‐6‐phosphate synthase gene (HvTPS) and investigated its expression patterns in various tissues and developmental stages in Heortia vitessoides Moore (Lepidoptera: Crambidae). HvTPS was highly expressed in the fat body and after pupation or before molting. We knocked down TPS in H. vitessoides by RNA interference and found that 3.0 μg of dsHvTPS resulted in optimal interference at 24 h and 36 h post‐injection and caused a sharp decline in the survival rate during the 5th instar larval–pupal stage and obviously abnormal or lethal phenotypes. Additionally, compared to the controls, TPS activity and trehalose contents were significantly lower and the glucose content was significantly higher 24 h or 36 h after injection with 3.0 μg of dsHvTPS. Furthermore, the silencing of HvTPS suppressed the expression of six key genes in the chitin biosynthesis pathway and one key gene related to lipid catabolism. The expression levels of two genes associated with lipid biosynthesis were upregulated. These results strongly suggest that HvTPS is essential for the normal growth and development of H. vitessoides and provide a reference for further studies of the utility of key genes involved in chitin and lipid biosynthesis for controlling insect development.

Abstract The insect midgut peritrophic membrane (PM) is a functional layer that protects insects against abrasive food particles and microorganism infection. Chitin is an essential component of the PM, and its synthesis is catalyzed by chitin synthase 2 (CHS2). CHS2 plays a unique role in chitin synthesis in the PM and thus represents a potential target for eco-friendly pesticides. The chitin synthase 2 gene (CfCHS2) of the rusty grain beetle, Cryptolestes ferrugineus (Stephens), was identified and evaluated. The full-length open reading frame of CfCHS2 was 4428 nucleotides long, encoding 1475 amino acids. Phylogenetic analysis revealed that CfCHS2 clearly clustered with the group of CHS2 sequences from other insects. Real-time quantitative PCR analysis showed that CfCHS2 was expressed in all developmental stages, with the highest expression level in the adult stage and the lowest in the pupal stage. In the adult stage, CfCHS2 was most highly expressed in the abdomen, followed by the midgut. RNAi assay results showed that the larvae curled up and could not crawl normally after CfCHS2 silencing. The midgut tissue of the larvae subjected to RNAi treatment was observed by tissue section scanner and transmission electron microscope (TEM). The results showed that the PM of the larvae was absent after CfCHS2 gene silencing. These results suggest that CfCHS2 is a potential RNAi target for C. ferrugineus control.

No abstract available

Chitin synthase (CHS) is the enzyme specifically associated with chitin synthesis, an important component of diverse organisms including insects. Two alternative spliced transcripts of the CHS gene (AsCHS-A1 and AsCHS-A2) were identified by quantitative reverse-transcription polymerase chain reaction (RT-qPCR) during the development of the leaf-cutting ant Atta sexdens. Expression profiles of AsCHS-A transcripts increased from larva to pupae and decay in workers. Phylogenetic analysis showed both transcripts are classified within class A insect CHSs. AsCHS-A1 showed the highest expression level in larvae and pupae, while AsCHS-A2 is the main CHS transcript in workers. Our results suggest that these variants should be under regulation of different promoters. AsCHS-A1 has topology expected for insect CHSs, while the predicted AsCHS-A2 topology, with a missing A domain, is similar to some fungal CHSs. CHS-B (class B) was not identified in A. sexdens transcriptome. Ribonucleic acid interference (RNAi)-mediated gene silencing in pupae revealed that low reduction in CHS transcript levels (18%) was enough to cause morphological changes in the pupa exoskeleton impairing the process of cuticle sclerotization. To our knowledge, this work was the first to use and to show the feasibility of using RNA interference techniques on leaf-cutting ants.

No abstract available

Chitin is an important part of the fungal cell wall, but is not found in plants and mammals, so chitin synthase (CHS) can be a green fungicide target. In this paper, 35 maleimide compounds were designed and synthesized as CHS inhibitors. All the screened compounds showed different degrees of CHS inhibitory activity and antifungal activity in vitro. In particular, the half–inhibitory concentration (IC50) value of compound 20 on CHS was 0.12 mM, and the inhibitory effect was better than that of the control polyoxin B (IC50 = 0.19 mM). At the same time, this compound also showed good antifungal activity and has further development value.

The development of multi‐action pesticides presents significant advantages, including cost‐effectiveness, reduced application frequency, enhanced resistance management, and minimized environmental impact. Despite its efficacy in targeting chitin synthase in both fungi and insects, the insecticidal performance of polyoxin B remains limited. To overcome this limitation, a novel nano‐formulation, polyoxin B@ZTS is developed, utilizing agricultural byproducts, tea saponin, and zein. This formulation features a small particle size, high leaf deposition efficiency, and excellent dispersion properties. Leveraging a disulfide bond system, polyoxin B@ZTS is designed to respond to the intracellular reducing environment, enabling controlled release of polyoxin B. Remarkably, the nano‐formulation facilitates the penetration of physiological barriers, achieving equivalent insecticidal and fungicidal efficacy to polyoxin B at only one‐fifth of the dosage. This work highlights the potential of eco‐friendly, cost‐effective, and multifunctional nano‐pesticides, providing a compelling solution for sustainable agricultural practices and demonstrating broad applicability in integrated pest and disease management.

No abstract available

Spodoptera exigua (Lepidoptera, Noctuidae) has been responsible for causing considerable and widespread agricultural losses worldwide. Owing to strong selective pressure, S. exigua showed increased resistance to Lufenuron (LUF). Consequently, RNA interference (RNAi)-based insecticides had more benefits than chemical insecticides. Therefore, to enhance the insecticidal activity of LUF to S. exigua, in the present study, we aimed to elucidate the impact of double-stranded RNAs (dsRNAs) on S. exigua larval susceptibility to LUF. First, the transcriptome of S. exigua was sequenced following the treatment with LUF. By comparing the upregulated and downregulated GO enrichment, chitin binding and chitin metabolic processes were the significantly enriched pathways. According to transcriptome sequencing, 8 genes associated with chitin biosynthesis, 8 chitin degradation genes, and 17 cuticle protein genes were obtained. UDP-N-acetylglucosamine pyrophosphorylase (UAP) and Chitin synthase A (CHSA) showed significantly downregulated expression after treatment with different sublethal doses of LUF. Downregulation of UAP increased mortality from 31.97% to 47.91% when the larvae were exposed to LUF. A significant increase in the mortality of S. exigua from 30.63% to 50.19% was observed following LUF administration after dsCHSA. In addition, the expression analysis of genes associated with chitin biosynthesis was significantly changed after LUF treatment, dsRNAs-RNAi, and their combination (LUF-dsRNAs). Significant differences were observed in the chitin content between the control group at 72 h after treatments. Results of the present study can help further elucidate the understanding of the combined effects of RNAi and LUF on S. exigua. Additionally, this research provides a suitable foundation for future studies with the aim to develop an efficient method of delivery for large-scale pest control in the fields.

Chitin synthase serves as a promising target for developing eco-friendly insecticides. Nevertheless, the lack of structural insights into insect chitin synthase has impeded the rational design of specific inhibitors. Herein, we utilized AlphaFold to predict the structure of PxChs1. A series of V-type amidazoles mimicking the catalytic sites of PxChs1 were synthesized. Notably, compounds 6k and 6l exhibited significant insecticidal efficacy against Plutella xylostella, with LC50 values of 0.789 and 0.951 μg/mL, respectively, and induced profound molting disruptions in the larvae. Moreover, the expression of genes involved in chitin metabolism was significantly downregulated. V-type amidazoles with potent insecticidal activity showed favorable docking interactions with PxChs1. Notably, compounds 6k and 6l exhibited minimal toxicity toward Chinese honeybees and Danio rerio. This study provides a valuable approach for developing innovative PxChs1 inhibitors despite a limited structural understanding of the protein.

We examined the susceptibility of field strains (BO-1, BO-2, TO-1, and YH-1) and one laboratory strain (H-1) of the western flower thrip, Frankliniella occidentalis, to benzoylureas. LC50 values of novaluron were determined as 0.64 ppm against laboratory strain and 2.1-130 ppm against field strains. In the presence of piperonyl butoxide, a cytochrome P450 inhibitor, the insecticidal activity of novaluron tended to be enhanced. To examine whether point mutations in chitin synthase 1 (CHS1) discovered in an etoxazole-resistant strain of Tetranychus urticae and a benzoylurea-resistant strain of Plutella xylostella exist in F. occidentalis, the nucleotide sequence of CHS1 was analyzed. We found a nonsynonymous substitution that corresponded to the location of the mutations found in T. urticae and P. xylostella in the field strains of F. occidentalis but not in the laboratory strain, indicating that this point mutation might be associated with the benzoylurea resistance exhibited by the field strains.

The small brown planthopper, Laodelphax striatellus, is a dangerous pest in rice fields. Although buprofezin has been used to control L. striatellus for more than a decade, the occurrence of buprofezin-resistant L. striatellus has been recently reported. To develop an alternative pest control strategy, comparative transcriptome analysis of buprofezin-treated and nontreated L. striatellus was performed to screen the buprofezin-specific target genes for RNA interference (RNAi) application. Among six genes downregulated in the buprofezin-treated L. striatellus, RNAi-based silencing of the lipophorin precursor, endocuticle structure glycoprotein, and chitin synthase significantly induced the lethality of L. striatellus in a concentration-dependent manner. In addition, a cocktail of double-stranded RNAs against these three genes showed synergistic effects with buprofezin. These results provide RNAi-based effective approaches to control L. striatellus as well as an efficient method to identify novel target genes for RNAi application.

Lufenuron is an effective benzoylurea insecticide that inhibits the synthesis of chitin and regulates the growth of insects. However, little is known about the effects of lufenuron treatment on the development of Spodoptera frugiperda (J. E. Smith). In this study, we assessed the toxicity of lufenuron on S. frugiperda and evaluated the effects of lufenuron treatment on the growth and development of S. frugiperda. The results showed that lufenuron exhibits high insecticidal activity against S. frugiperda, with the LC50 value of 0.99 mg L-1. Lufenuron treatments can significantly prolong the larval developmental duration and reduce the rates of pupation and emergence. To further explore the underlying mechanism of this observation, the expression profiles of the chitin synthase gene (SfCHS) and chitinase gene (SfCHT), two key enzyme genes involved in the molting of S. frugiperda, were determined after exposure to lufenuron for 96 h. The results of qRT-PCR demonstrated that lufenuron treatments can significantly reduce the expression of SfCHT, while the expression of SfCHS remained relatively stable. Furthermore, we found that lufenuron strongly interacted with chitinase (SfCHT) (-10.8 kcal/mol) and chitin synthase (SfCHS) (R1: -9.7 kcal/mol; R2: -10.2 kcal/mol). Our results indicated that lufenuron has significant effects on the development of S. frugiperda that might be attributed to the differential expression of SfCHT and SfCHS.

Among the six-membered heterocycles, the pyrazine ring is poorly explored in crop protection and does not feature in any product listed in the current IRAC MoA classification. In an effort to identify new leads for internal research, we synthesized a series of N-(5-phenylpyrazin-2-yl)-benzamide derivatives and evaluated them for their insecticidal activity. N-(5-phenylpyrazin-2-yl)-benzamide derivatives 3 were prepared using an automated two-step synthesis protocol. These compounds were tested for their initial biological activity against a wide range of sucking and chewing insect pests and found to be active against lepidopterans only. More detailed experiments, including symptomology studies on the diamondback moth, Plutella xylostella (L.) and the Egyptian cotton leafworm, Spodoptera littoralis (Boisduval) showed that analog 3q causes severe abnormalities in the lepidopteran cuticle leading to larval mortality. Compound 3q shows strong potency against both P. xylostella and S. littoralis, whereas analog 3i shows better potency against S. littoralis causing also impaired cuticular structure and death of the larvae. Additionally, P. xylostella genetic studies showed that compound 3q resistance is linked to Chitin Synthase 1. Our studies show that N-(5-phenylpyrazin-2-yl)-benzamide derivatives 3, and in particular analogs 3i and 3q, act as insect growth modulator insecticides. Conformational similarities with lufenuron are discussed.

Tetranychus urticae, a highly destructive global pest, infests a wide range of plant species, including crucial food crops and ornamental plants. Effective control methods for this pest remain limited. Chitin synthase (CHS) is a key enzyme in the biosynthesis of chitin, which is essential for the growth and development of arthropods. However, the lack of detailed research on arthropod CHS proteins has hindered the development of targeted pesticides. In this study, we successfully expressed and purified the full-length TuCHS protein, which exhibited significant enzymatic activity in vitro. Utilizing this protein, we developed a reliable screening method to identify inhibitors targeting TuCHS. Two inhibitors, ZHZ-ZI-11 and SUY-SC-15, were identified. These compounds interfere with chitin translocation within the cell rather than inhibiting CHS enzyme activity. Both inhibitors demonstrated significant acaricidal efficacy, with improved performance when formulated as nanoemulsions. This study presents the first use of arthropod CHS protein for screening potential insecticides targeting chitin biosynthesis during mite development. Our findings provide a solid foundation for the development of novel, environmentally friendly pesticides aimed at CHS.