Plasticity in moth mating behavior and ejaculate proteomic diversity induced by male competition.

In male competition, large and costly ejaculates are advantageous. Prior research on male accessory gland secretions in Plutella xylostella left open questions about how males modulate their mating behaviors and ejaculate composition allocation in response to varying levels of competition. The current study aimed to delve deeper into these unexplored facets. A totally of 928 ejaculate proteins were identified across males exposed to different competition conditions. Notably, males courting under non-, low-, and high-competition scenarios exhibited 867, 635, and 858 ejaculate proteins, respectively. Approximately 10% of these ejaculate proteins displayed variations that aligned with changes in competition intensity. Subsequent analyses focused on the proteins transferred to females, revealing that 44% of ejaculate proteins were transferred, with 37 proteins exhibiting differential expression. Functional analyses uncovered their crucial roles in sperm maturation, motility, and capacitation. Our findings reveal adaptive adjustments in ejaculate protein abundance and transmission in P. xylostella as a response to varying competition levels. Moreover, fluorescent sperm labeling indicated higher sperm transfer during low competition correlated with shorter sperm length. Furthermore, evidence suggests that males shorten their courtship duration and extend their mating duration when faced with competition. These results illustrate how competition drives ejaculate investment and behavioral plasticity, offering valuable insights for advancements in assisted reproductive technologies and pest management strategies.

Trehalase inhibition in Helicoverpa armigera activates machinery for alternate energy acquisition.

Trehalose serves as a primary circulatory sugar in insects which is crucial in energy metabolism and stress recovery. It is hydrolyzed into two glucose molecules by trehalase. Silencing or inhibiting trehalase results in reduced fitness, developmental defects, and insect mortality. Despite its importance, the molecular response of insects to trehalase inhibition is not known. Here, we performed transcriptomic analyses of Helicoverpa armigera treated with validamycin A (VA), a trehalase inhibitor. VA ingestion resulted in increased mortality, developmental delay, and reduced ex vivo trehalase activity. Pathway enrichment and gene ontology analyses suggest that key genes involved in carbohydrate, protein, fatty acid, and mitochondria-related metabolisms are deregulated. The activation of protein and fat degradation may be necessary to fulfil energy requirements, evidenced by the dysregulated expression of critical genes in these metabolisms. Co-expression analysis supports the notion that trehalase inhibition leads to putative interaction with key regulators of other pathways. Metabolomics correlates with transcriptomics to show reduced levels of key energy metabolites. VA generates an energy-deficient condition, and insects activate alternate pathways to facilitate the energy demand. Overall, this study provides insights into the molecular mechanisms underlying the response of insects to trehalase inhibition and highlights potential targets for insect control.

Four New Furofuran Lignans from Phryma leptostachya Inhibit the Accumulation of Molting Hormones in Armyworm.

Furofuran lignans have been identified as the main substances responsible for the biological activities of the plant genus Phryma. Here, four new phrymarolin-type leptolignans A-D (7-10) and eight previously known lignans were isolated from P. leptostachya. Of these, nine exhibited significant antifeedant activity against armyworm (Mythimna separata) through a dual-choice bioassay, with the EC50 values ranging from 0.58 to 10.08 µg/cm2. In particular, the newly identified lignan leptolignan A (7) showed strong antifeedant activity, with an EC50 value of 0.58 ± 0.34 µg/cm2. Further investigation found that leptolignan A can inhibit the growth and nutritional indicators in the armyworm M. separata. The concentrations of two molting hormones, 20-hydroxyecdysone and ecdysone, were also found to decrease significantly following the treatment of the armyworms with the lignan, implying that the target of the P. leptostachya lignan may be involved in 20-hydroxyecdysone and ecdysone synthesis. These results enrich our knowledge of P. leptostachya metabolite structural diversity, and provide a theoretical basis for the control of armyworm using lignans.

Molecular Basis of CO2 Sensing in Hyphantria cunea.

Carbon dioxide (CO2) released by plants can serve as a cue for regulating insect behaviors. Hyphantria cunea is a widely distributed forestry pest that may use CO2 as a cue for foraging and oviposition. However, the molecular mechanism underlying its ability to sense CO2 has not been elucidated. Our initial study showed that CO2 is significantly attractive to H. cunea adults. Subsequently, 44 H. cunea gustatory receptors (GRs) were identified using transcriptome data, and 3 candidate CO2 receptors that are specifically expressed in the labial palps were identified. In vivo electrophysiological assays revealed that the labial palp is the primary organ for CO2 perception in H. cunea, which is similar to findings in other lepidopteran species. By using the Xenopus oocyte expression system, we showed that the HcunGR1 and HcunGR3 co-expressions produced a robust response to CO2, but HcunGR2 had an inhibitory effect on CO2 perception. Finally, immunohistochemical staining revealed sexual dimorphism in the CO2-sensitive labial pit organ glomerulus (LPOG). Taken together, our results clarified the mechanism by which H. cunea sense CO2, laying the foundation for further investigations into the role of CO2 in the rapid spread of H. cunea.

Ecotoxicity of Cadmium along the Soil-Cotton Plant-Cotton Bollworm System: Biotransfer, Trophic Accumulation, Plant Growth, Induction of Insect Detoxification Enzymes, and Immunocompetence.

Cadmium (Cd) is a hazardous element that may jeopardize environmental safety and human health through biotransfer and trophic accumulation. Here, we tested Cd toxicity on cotton plants, cotton bollworms, and their responses. Results demonstrated that Cd accumulated in plant roots, aerial parts, insect larvae, pupae, and frass in a dose-dependent pattern. The ∼9.35 mg kg-1 of Cd in plant aerial parts, ∼3.68 in larvae, ∼6.43 in pupae, and high transfer coefficient (∼5.59) indicate significant mobility. The ∼19.61 mg kg-1 of Cd in larvae frass suggests an effective detoxification strategy, while BAFcotton (∼1.14) and BAFworm (∼0.54) indicated low bioaccumulation. Cadmium exposure resulted in compromised plant growth and yield as well as alterations in photosynthetic pigment contents, antioxidant enzyme activities, and certain life history traits of cotton bollworms. Furthermore, carboxylesterase activity and encapsulation rates of insect larvae decreased with increasing Cd concentrations, whereas acetylcholinesterase, phenol oxidase, glutathione S-transferase, and multifunctional oxidase exhibited hormesis responses.

The Key Role of Plant Hormone Signaling Transduction and Flavonoid Biosynthesis Pathways in the Response of Chinese Pine (Pinus tabuliformis) to Feeding Stimulation by Pine Caterpillar (Dendrolimus tabulaeformis).

In nature, plants have developed a series of resistance mechanisms to face various external stresses. As understanding of the molecular mechanisms underlying plant resistance continues to deepen, exploring endogenous resistance in plants has become a hot topic in this field. Despite the multitude of studies on plant-induced resistance, how plants respond to stress under natural conditions remains relatively unclear. To address this gap, we investigated Chinese pine (Pinus tabuliformis) using pine caterpillar (Dendrolimus tabulaeformis) under natural conditions. Healthy Chinese pine trees, approximately 10 years old, were selected for studying induced resistance in Huangtuliangzi Forestry, Pingquan City, Chengde City, Hebei Province, China. Pine needles were collected at 2 h and 8 h after feeding stimulation (FS) via 10 pine caterpillars and leaf clipping control (LCC), to simulate mechanical damage caused by insect chewing for the quantification of plant hormones and transcriptome and metabolome assays. The results show that the different modes of treatments significantly influence the contents of JA and SA in time following treatment. Three types of differentially accumulated metabolites (DAMs) were found to be involved in the initial response, namely phenolic acids, lipids, and flavonoids. Weighted gene co-expression network analysis indicated that 722 differentially expressed genes (DEGs) are positively related to feeding stimulation and the specific enriched pathways are plant hormone signal transduction and flavonoid biosynthesis, among others. Two TIFY transcription factors (PtTIFY54 and PtTIFY22) and a MYB transcription factor (PtMYB26) were found to be involved in the interaction between plant hormones, mainly in the context of JA signal transduction and flavonoid biosynthesis. The results of this study provide an insight into how JA activates, serving as a reference for understanding the molecular mechanisms of resistance formation in conifers responding to mandibulate insects.

The novel function of an orphan pheromone receptor reveals the sensory specializations of two potential distinct types of sex pheromones in noctuid moth.

Sex pheromones play crucial role in mating behavior of moths, involving intricate recognition mechanisms. While insect chemical biology has extensively studied type I pheromones, type II pheromones remain largely unexplored. This study focused on Helicoverpa armigera, a representative species of noctuid moth, aiming to reassess its sex pheromone composition. Our research unveiled two previously unidentified candidate type II sex pheromones-3Z,6Z,9Z-21:H and 3Z,6Z,9Z-23:H-in H. armigera. Furthermore, we identified HarmOR11 as an orphan pheromone receptor of 3Z,6Z,9Z-21:H. Through AlphaFold2 structural prediction, molecular docking, and molecular dynamics simulations, we elucidated the structural basis and key residues governing the sensory nuances of both type I and type II pheromone receptors, particularly HarmOR11 and HarmOR13. This study not only reveals the presence and recognition of candidate type II pheromones in a noctuid moth, but also establishes a comprehensive structural framework for PRs, contributing to the understanding of connections between evolutionary adaptations and the emergence of new pheromone types.

Dissecting the manipulation of lufenuron on chitin synthesis in Helicoverpa armigera.

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.

Functional study on candidate regulators mediating the expression of CYP6B7 induced by fenvalerate in a susceptible strain of Helicoverpa armigera.

Transcription factors play an important role in regulating the expression of detoxification genes (e.g. P450s) that confer insecticide resistance. Our previous study identified a series of candidate transcription factors (CYP6B7-fenvalerate association proteins, CAPs) that may be related to fenvalerate-induced expression of CYP6B7 in a field HDTJ strain of H. armigera. Whether these CAPs can mediate the transcript of CYP6B7 induced by fenvalerate in a susceptible HDS strain of H. armigera remains unknown. Further study showed that the expression levels of multiple CAPs were significantly induced by fenvalerate in HDS strain. Knockdown of CAP19 [fatty acid synthase-like (FAS)], CAP22 [polysaccharide biosynthesis domain-containing protein 1 (PBDC1)], CAP24 [5-formyltetrahydrofolate cycloligase (5-FCL)], CAP30 [peptidoglycan recognition protein LB-like (PGRP)] and CAP33 [NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 11 (NDUFA11)] resulted in significant inhibition of CYP6B7 and some other P450 genes expression; meanwhile, the sensitivity of HDS strain larvae to fenvalerate was significantly increased. In addition, PBDC1, PGRP and NDUFA11, either alone or in combination, could significantly enhance the activity of CYP6B7 promoter in HDS strain, as well as the expression level of CYP6B7 gene in Sf9 cells line. These results suggested that PBDC1, PGRP and NDUFA11 may be involved in the transcript regulation of key detoxifying genes in response to fenvalerate in HDS strain of H. armigera.

No evidence for the melanin desiccation hypothesis in a larval Lepidopteran.

Water regulation is an important physiological challenge for insects due to their small body sizes and large surface area to volume ratios. Adaptations for decreasing cuticular water loss, the largest avenue of loss, are especially important. The melanin desiccation hypothesis states that melanin molecules in the cuticle may help prevent water loss, thus offering protection from desiccation. This hypothesis has much empirical support in Drosophila species, but remains mostly untested in other taxa, including Lepidoptera. Because melanin has many other important functions in insects, its potential role in desiccation prevention is not always clear. In this study we investigated the role of melanin in desiccation prevention in the white-lined Sphinx moth, Hyles lineata (Lepidoptera, Sphingidae), which shows high plasticity in the degree of melanin pigmentation during the late larval instars. We took advantage of this plasticity and used density treatments to induce a wide range of cuticular melanization; solitary conditions induced low melanin pigmentation while crowded conditions induced high melanin pigmentation. We tested whether more melanic larvae from the crowded treatment were better protected from desiccation in three relevant responses: i) total water loss over a desiccation period, ii) change in hemolymph osmolality over a desiccation period, and iii) evaporation rate of water through the cuticle. We did not find support for the melanin desiccation hypothesis in this species. Although treatment influenced total water loss, this effect did not occur via degree of melanization. Interestingly, this implies that crowding, which was used to induce high melanin phenotypes, may have other physiological effects that influence water regulation. There were no differences between treatments in cuticular evaporative water loss or change in hemolymph osmolality. However, we conclude that osmolality may not sufficiently reflect water loss in this case. This study emphasizes the context dependency of melanin's role in desiccation prevention and the importance of considering how it may vary across taxa. In lepidopteran larvae that are constantly feeding phytophagous insects with soft cuticles, melanin may not be necessary for preventing cuticular water loss.

A comprehensive gene expression analysis of the unique three-layered cocoon of the cecropia moth, Hyalophora cecropia.

The larvae of the moth Hyalophora cecropia spin silk cocoons with morphologically distinct layers. We investigated the expression of the individual silk protein components of these cocoons in relation to the morphology of the silk gland and its affiliation to the different layers of the cocoon. The study used transcriptomic and proteomic analyses to identify 91 proteins associated with the silk cocoons, 63 of which have a signal peptide indicating their secretory nature. We checked the specificity of their expression in different parts of the SG and the presence of the corresponding protein products in each cocoon layer. Differences were observed among less abundant proteins with unclear functions. The representation of proteins in the inner envelope and intermediate space was similar, except for a higher proportion of probable contaminating proteins, mostly originating from the gut. On the other hand, the outer envelope contains a number of putative enzymes with unclear function. However, the protein most specific to the outer layer has sequence homology to putative serine/threonine kinase-like proteins and some adhesive proteins, and its closest homolog in Bombyx mori was found in the scaffold silk. This research provides valuable insights into the silk production of the cecropia moth, highlighting both similarities and differences to other moth species.

The role of a novel secretory peptidoglycan recognition protein with antibacterial ability from the Chinese Oak Silkworm Antheraea pernyi in humoral immunity.

Peptidoglycan recognition proteins (PGRPs) are a family of pattern recognition receptors that play a critical role in the immune response of invertebrates and vertebrates. Herein, the short ApPGRP-D gene was cloned from the model lepidopteran Antheraea pernyi. Quantitative PCR (qPCR) confirmed that ApPGRP-D is an immune-related protein and that the expression of ApPGRP-D can be induced by microorganisms. ApPGRP-D is a broad-spectrum pattern recognition protein that activates the prophenoloxidase cascade activation system and promotes the agglutination of microbial cells. Likely due to its amidase activity, ApPGRP-D can inhibit the growth of E. coli and S. aureus. In addition, we demonstrated for the first time that zinc ions, as important metal coenzymes, could promote multiple functions of ApPGRP-D but not its amidase activity.

Genome-wide profiles of H3K9me3, H3K27me3 modifications, and DNA methylation during diapause of Asian corn borer (Ostrinia furnacalis).

Diapause represents a crucial adaptive strategy used by insects to cope with changing environmental conditions. In North China, the Asian corn borer (Ostrinia furnacalis) enters a winter larval diapause stage. Although there is growing evidence implicating epigenetic mechanisms in diapause regulation, it remains unclear whether dynamic genome-wide profiles of epigenetic modifications exist during this process. By investigating multiple histone modifications, we have discovered the essential roles of H3K9me3 and H3K27me3 during diapause of the Asian corn borer. Building upon previous findings in vertebrates highlighting the connection between DNA methylation and repressive histone methylations, we have examined changes in the genome-wide profile of H3K9me3, H3K27me3, and DNA methylation at the nondiapause, prediapause, and diapause stages. Data analysis reveals significant alterations in these three modifications during diapause. Moreover, we observe a correlation between the H3K9me3 and H3K27me3 modification sites during diapause, whereas DNA modifications show little association with either H3K9me3 or H3K27me3. Integrative analysis of epigenome and expression data unveils the relationship between these epigenetic modifications and gene expression levels at corresponding diapause stages. Furthermore, by studying the function of histone modifications on genes known to be important in diapause, especially those involved in the juvenile pathway, we discover that the juvenile hormone pathway lies downstream from H3K9me3 and H3K27me3 histone modifications. Finally, the analysis of gene loci with modified modifications unreported in diapause uncovers novel pathways potentially crucial in diapause regulation. This study provides a valuable resource for future investigations aiming to elucidate the underlying mechanisms of diapause.

Ontogenetic variation in metabolic rate-temperature relationships during larval development.

Predictive models of ectotherm responses to environmental change often rely on thermal performance data from the literature. For insects, the majority of these data focus on two traits, development rate and thermal tolerance limits. Data are also often limited to the adult stage. Consequently, predictions based on these data generally ignore other measures of thermal performance and do not account for the role of ontogenetic variation in thermal physiology across the complex insect life cycle. Theoretical syntheses for predicting metabolic rate also make similar assumptions despite the strong influence of body size as well as temperature on metabolic rate. The aim of this study was to understand the influence of ontogenetic variation on ectotherm physiology and its potential impact on predictive modeling. To do this, we examined metabolic rate-temperature (MR-T) relationships across the larval stage in a laboratory strain of the spongy moth (Lymantria dispar dispar). Routine metabolic rates (RMRs) of larvae were assayed at eight temperatures across the first five instars of the larval stage. After accounting for differences in body mass, larval instars showed significant variation in MR-T. Both the temperature sensitivity and allometry of RMR increased and peaked during the third instar, then declined in the fourth and fifth instar. Generally, these results show that insect thermal physiology does not remain static during larval ontogeny and suggest that ontogenetic variation should be an important consideration when modeling thermal performance.

The Circadian Clock Gene PHYTOCLOCK1 Mediates the Diurnal Emission of the Anti-Insect Volatile Benzyl Nitrile from Damaged Tea (Camellia sinensis) Plants.

Benzyl nitrile from tea plants attacked by various pests displays a diurnal pattern, which may be closely regulated by the endogenous circadian clock. However, the molecular mechanism by the circadian clock of tea plants that regulates the biosynthesis and release of volatiles remains unclear. In this study, the circadian clock gene CsPCL1 can activate both the expression of the benzyl nitrile biosynthesis-related gene CsCYP79 and the jasmonic acid signaling-related transcription factor CsMYC2 involved in upregulating CsCYP79 gene, thereby resulting in the accumulation and release of benzyl nitrile. Therefore, the anti-insect function of benzyl nitrile was explored in the laboratory. The application of slow-release beads of benzyl nitrile in tea plantations significantly reduced the number of tea geometrids and had positive effects on the yield of fresh tea leaves. These findings reveal the potential utility of herbivore-induced plant volatiles for the green control of pests in tea plantations.

Characterization of the individual domains of the Bacillus thuringiensis Cry2Aa implicates Domain I as a possible binding site to Helicoverpa armigera.

Bacillus thuringiensis (Bt) Cry2Aa is a member of the Cry pore-forming, 3-domain, toxin family with activity against both lepidopteran and dipteran insects. Although domains II and III of the Cry toxins are believed to represent the primary specificity determinant through specific binding to cell receptors, it has been proposed that the pore-forming domain I of Cry2Aa also has such a role. Thus, a greater understanding of the functions of Cry2Aa's different domains could potentially be helpful in the rational design of improved toxins. In this work, cry2Aa and its domain fragments (DI, DII, DIII, DI-II and DII-DIII) were subcloned into the vector pGEX-6P-1 and expressed in Escherichia coli. Each protein was recognized by anti-Cry2Aa antibodies and, except for the DII fragment, could block binding of the antibody to Cry2Aa. Cry2Aa and its DI and DI-II fragments bound to brush border membrane vesicles (BBMV) from H. armigera and also to a ca 150 kDa BBMV protein on a far western (ligand) blot. In contrast the DII, DIII and DII-III fragments bound to neither of these. None of the fragments were stable in H. armigera gut juice nor showed any toxicity towards this insect. Our results indicate that contrary to the general model of Cry toxin activity domain I plays a role in the binding of the toxin to the insect midgut.

Functional Analysis of Insecticide Inhibition and Metabolism of Six Glutathione S-Transferases in the Rice Stem Borer, Chilo suppressalis.

The glutathione S-transferases (GSTs) are important detoxifying enzymes in insects. Our previous studies found that the susceptibility of Chilo suppressalis to abamectin was significantly increased when the CsGST activity was inhibited by glutathione (GSH) depletory. In this study, the potential detoxification mechanisms of CsGSTs to abamectin were explored. Six CsGSTs of C. suppressalis were expressed in vitro. Enzymatic kinetic parameters including Km and Vmax of recombinant CsGSTs were determined, and results showed that all of the six CsGSTs were catalytically active and displaying glutathione transferase activity. Insecticide inhibitions revealed that a low concentration of abamectin could effectively inhibit the activities of CsGSTs including CsGSTd1, CsGSTe4, CsGSTo2, CsGSTs3, and CsGSTu1. However, the in vitro metabolism assay found that the six CsGSTs could not metabolize abamectin directly. Additionally, the glutathione transferase activity of CsGSTs in C. suppressalis was significantly increased post-treatment with abamectin. Comprehensive analysis of the results in present and our previous studies demonstrated that CsGSTs play an important role in detoxification of abamectin by catalyzing the conjugation of GSH to abamectin in C. suppressalis, and the high binding affinities of CsGSTd1, CsGSTe4, CsGSTo2, CsGSTs3, and CsGSTu1 with abamectin might also suggest the involvement of CsGSTs in detoxification of abamectin via the noncatalytic passive binding and sequestration instead of direct metabolism. These studies are helpful to better understand the detoxification mechanisms of GSTs in insects.

A chromosome-level genome assembly of the soybean pod borer: insights into larval transcriptional response to transgenic soybean expressing the pesticidal Cry1Ac protein.

BACKGROUND: Genetically modified (GM) crop plants with transgenic expression of Bacillus thuringiensis (Bt) pesticidal proteins are used to manage feeding damage by pest insects. The durability of this technology is threatened by the selection for resistance in pest populations. The molecular mechanism(s) involved in insect physiological response or evolution of resistance to Bt is not fully understood. RESULTS: To investigate the response of a susceptible target insect to Bt, the soybean pod borer, Leguminivora glycinivorella (Lepidoptera: Tortricidae), was exposed to soybean, Glycine max, expressing Cry1Ac pesticidal protein or the non-transgenic parental cultivar. Assessment of larval changes in gene expression was facilitated by a third-generation sequenced and scaffolded chromosome-level assembly of the L. glycinivorella genome (657.4 Mb; 27 autosomes + Z chromosome), and subsequent structural annotation of 18,197 RefSeq gene models encoding 23,735 putative mRNA transcripts. Exposure of L. glycinivorella larvae to transgenic Cry1Ac G. max resulted in prediction of significant differential gene expression for 204 gene models (64 up- and 140 down-regulated) and differential splicing among isoforms for 10 genes compared to unexposed cohorts. Differentially expressed genes (DEGs) included putative peritrophic membrane constituents, orthologs of Bt receptor-encoding genes previously linked or associated with Bt resistance, and those involved in stress responses. Putative functional Gene Ontology (GO) annotations assigned to DEGs were significantly enriched for 36 categories at GO level 2, respectively. Most significantly enriched cellular component (CC), biological process (BP), and molecular function (MF) categories corresponded to vacuolar and microbody, transport and metabolic processes, and binding and reductase activities. The DEGs in enriched GO categories were biased for those that were down-regulated (≥ 0.783), with only MF categories GTPase and iron binding activities were bias for up-regulation genes. CONCLUSIONS: This study provides insights into pathways and processes involved larval response to Bt intoxication, which may inform future unbiased investigations into mechanisms of resistance that show no evidence of alteration in midgut receptors.

Molecular characterization and functional analysis of the ecdysone receptor isoform (EcR) from the oriental fruit moth Grapholita molesta (Lepidoptera: Tortricidae).

20-Hydroxyecdysone (20E) plays a vital role in a series of biological processes, via the nuclear receptors, EcR/USP by activating the ecdysone regulatory cascade. To clarify the role of EcR during the development of Grapholita molesta, the complementary DNA of ecdysone receptor isoform B1 (GmEcR-B1) was obtained from the transcriptome of G. molesta and verified by PCR. Alignment analysis revealed that the deduced protein sequence of GmEcR-B1 was highly homologous to EcR proteins identified in other lepidopteran species, especially the EcR-B1 isoform in Spodoptera litura. Quantitative real-time PCR showed that GmEcRs was expressed at all test developmental stages, and the expression level of GmEcRs was relatively higher during the period of the 3rd day of fifth instar larvae to 2nd of pupa than those in other stages. Moreover, the messenger RNA of GmEcRs was much more strongly expressed in the Malpighian tubule and epidermis than those in other tissues, which suggests that this gene may function in a tissue-specific manner during larval development. Silencing of GmEcRs could significantly downregulate the transcriptional level of ecdysone-inducible genes and result in increased mortality during metamorphosis and prolonged prepupal duration. Taken together, the present results indicate that GmEcRs may directly or indirectly affect the development of G. molesta.

A Potential Multitarget Insect Growth Regulator Candidate: Design, Synthesis, and Biological Activity of Novel Acetamido Derivatives Containing Hexacyclic Pyrazole Carboxamides.

Insect growth regulators (IGRs) are important green insecticides that disrupt normal growth and development in insects to reduce the harm caused by pests to crops. The ecdysone receptor (EcR) and three chitinases OfChtI, OfChtII, and OfChi-h are closely associated with the molting stage of insects. Thus, they are considered promising targets for the development of novel insecticides such as IGRs. Our previous work identified a dual-target compound 6j, which could act simultaneously on both EcR and OfChtI. In the present study, 6j was first found to have inhibitory activities against OfChtII and OfChi-h, too. Subsequently, taking 6j as a lead compound, 19 novel acetamido derivatives were rationally designed and synthesized by introducing an acetamido moiety into the amide bridge based on the flexibility of the binding cavities of 6j with EcR and three chitinases. Then, their insecticidal activities against Plutella xylostella (P. xylostella), Ostrinia furnacalis (O. furnacalis), and Spodoptera frugiperda (S. frugiperda) were carried out. The bioassay results revealed that most of these acetamido derivatives possessed moderate to good larvicidal activities against three lepidopteran pests. Especially, compound I-17 displayed excellent insecticidal activities against P. xylostella (LC50, 93.32 mg/L), O. furnacalis (LC50, 114.79 mg/L), and S. frugiperda (86.1% mortality at 500 mg/L), significantly better than that of 6j. In addition, further protein validation and molecular docking demonstrated that I-17 could act simultaneously on EcR (17.7% binding activity at 8 mg/L), OfChtI (69.2% inhibitory rate at 50 µM), OfChtII (71.5% inhibitory rate at 50 µM), and OfChi-h (73.9% inhibitory rate at 50 µM), indicating that I-17 is a potential lead candidate for novel multitarget IGRs. This work provides a promising starting point for the development of novel types of IGRs as pest management agents.