CRISPR/Cas9-Mediated Knockout of the PxJHBP Gene Resulted in Increased Susceptibility to Bt Cry1Ac Protoxin and Reduced Lifespan and Spawning Rates in Plutella xylostella.

Juvenile hormone binding protein (JHBP) is a key regulator of JH signaling, and crosstalk between JH and 20-hydroxyecdysone (20E) can activate and fine-tune the mitogen-activated protein kinase cascade, leading to resistance to insecticidal proteins from Bacillis thuringiensis (Bt). However, the involvement of JHBP in the Bt Cry1Ac resistance of Plutella xylostella remains unclear. Here, we cloned a full-length cDNA encoding JHBP, and quantitative real-time PCR (qPCR) analysis showed that the expression of the PxJHBP gene in the midgut of the Cry1Ac-susceptible strain was significantly higher than that of the Cry1Ac-resistant strain. Furthermore, CRISPR/Cas9-mediated knockout of the PxJHBP gene significantly increased Cry1Ac susceptibility, resulting in a significantly shorter lifespan and reduced fertility. These results demonstrate that PxJHBP plays a critical role in the resistance to Cry1Ac protoxin and in the regulation of physiological metabolic processes associated with reproduction in adult females, providing valuable insights to improve management strategies of P. xylostella.

Electrophysiological and pharmacological properties of the slowpoke channel in the diamondback moth, Plutella xylostella.

The slowpoke channel responds to the intracellular calcium concentration and the depolarization of the cell membrane. It plays an important role in maintaining the resting potential and regulating the homeostasis of neurons, but it can also regulate circadian rhythm, sperm capacitation, ethanol tolerance, and other physiological processes in insects. This renders it a potentially useful target for the development of pest control strategies. There are relatively few studies on the slowpoke channels in lepidopteran pests, and their pharmacological properties are still unclear. So, in this study, the slowpoke gene of Plutella xylostella (Pxslo) was heterologous expressed in HEK293T cells, and the I-V curve of the slowpoke channel was measured by whole cell patch clamp recordings. Results showed that the slowpoke channel could be activated at -20 mV with 150 µM Ca2+. The subsequent comparison of the electrophysiological characteristics of the alternative splicing site E and G deletions showed that the deletion of the E site enhances the response of the slowpoke channel to depolarization, while the deletion of the G site weakens the response of the slowpoke channel to depolarization. Meanwhile, the nonspecific inhibitors TEA and 4-AP of the Kv channels, and four pesticides were tested and all showed an inhibition effect on the PxSlo channel at 10 or 100 µM, suggesting that these pesticides also target the slowpoke channel. This study enriches our understanding of the slowpoke channel in Lepidopteran insects and can aid in the development of relevant pest management strategies.

Tyrosine hydroxylase plays crucial roles in larval cuticle formation and larval-pupal tanning in the rice stem borer, Chilo suppressalis.

The striped stem borer, Chilo suppressalis (Walker), a notorious pest infesting rice, has evolved a high level of resistance to many commonly used insecticides. In this study, we investigate whether tyrosine hydroxylase (TH), which is required for larval development and cuticle tanning in many insects, could be a potential target for the control of C. suppressalis. We identified and characterized the full-length cDNA (CsTH) of C. suppressalis. The complete open reading frame of CsTH (MW690914) was 1683 bp in length, encoding a protein of 560 amino acids. Within the first to the sixth larval instars, CsTH was high in the first day just after molting, and lower in the ensuing days. From the wandering stage to the adult stage, levels of CSTH began to rise and reached a peak at the pupal stage. These patterns suggested a role for the gene in larval development and larval-pupal cuticle tanning. When we injected dsCsTH or 3-iodotyrosine (3-IT) as a TH inhibitor or fed a larva diet supplemented with 3-IT, there were significant impairments in larval development and larval-pupal cuticle tanning. Adult emergence was severely impaired, and most adults died. These results suggest that CsTH might play a critical role in larval development as well as larval-pupal tanning and immunity in C. suppressalis, and this gene could form a potential novel target for pest control.

Discovery of new N-Phenylamide Isoxazoline derivatives with high insecticidal activity and reduced honeybee toxicity.

Isoxazoline is a novel structure with strong potential for controlling agricultural insect pests, but its high toxicity to honeybees limits its development in agriculture. Herein, a series of N-phenylamide isoxazoline derivatives with low honeybee toxicity were designed and synthesized using the intermediate derivatization method. Bioassay results showed that these compounds exhibited good insecticidal activity. Compounds 3b and 3f showed significant insecticidal effects against Plutella xylostella (P. xylostella) with median lethal concentrations (LC50) of 0.06 and 0.07 mg/L, respectively, comparable to that of fluralaner (LC50 = 0.02 mg/L) and exceeding that of commercial insecticide fluxametamide (LC50 = 0.52 mg/L). It is noteworthy that the acute honeybee toxicities of compounds 3b and 3f (LD50 = 1.43 and 1.63 µg/adult, respectively) were significantly reduced to 1/10 of that of fluralaner (LD50 = 0.14 µg/adult), and were adequate or lower than that of fluxametamide (LD50 = 1.14 µg/adult). Theoretical simulation using molecular docking indicates that compound 3b has similar binding modes with fluralaner and a similar optimal docking pose with fluxametamide when binding to the GABA receptor, which may contribute to its potent insecticidal activity and relatively low toxicity to honey bees. This study provides compounds 3b and 3f as potential new insecticide candidates and provides insights into the development of new isoxazoline insecticides exhibiting both high efficacy and environmental safety.

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.

Prolonged Heat Stress during Winter Diapause Alters the Expression of Stress-Response Genes in Ostrinia nubilalis (Hbn.).

During diapause, a state of temporarily arrested development, insects require low winter temperatures to suppress their metabolism, conserve energy stores and acquire cold hardiness. A warmer winter could, thus, reduce diapause incidence and duration in many species, prematurely deplete their energy reserves and compromise post-diapause fitness. In this study, we investigated the combined effects of thermal stress and the diapause program on the expression of selected genes involved in antioxidant defense and heat shock response in the European corn borer Ostrinia nubilalis. By using qRT-PCR, it has been shown that response to chronic heat stress is characterized by raised mRNA levels of grx and trx, two important genes of the antioxidant defense system, as well as of hsp70 and, somewhat, of hsp90, two major heat shock response proteins. On the other hand, the expression of hsc70, hsp20.4 and hsp20.1 was discontinuous in the latter part of diapause, or was strongly controlled by the diapause program and refractory to heat stress, as was the case for mtn and fer, genes encoding two metal storage proteins crucial for metal ion homeostasis. This is the first time that the effects of high winter temperatures have been assessed on cold-hardy diapausing larvae and pupae of this important corn pest.

Discovery of Trisubstituted N-Phenylpyrazole Containing Diamides with Improved Insecticidal Activity.

To increase the structural diversity of insecticides and meet the needs of effective integrated insect management, the structure of chlorantraniliprole was modified based on a previously established three-dimensional quantitative structure-activity relationship (3D-QSAR) model. The pyridinyl moiety in the structure of chlorantraniliprole was replaced with a 4-fluorophenyl group. Further modifications of this 4-fluorophenyl group by introducing a halogen atom at position 2 and an electron-withdrawing group (e.g., iodine, cyano, and trifluoromethyl) at position 5 led to 34 compounds with good insecticidal efficacy against Mythimna separata, Plutella xylostella, and Spodoptera frugiperda. Among them, compound IV f against M. separata showed potency comparable to that of chlorantraniliprole. IV p against P. xylostella displayed a 4.5 times higher potency than chlorantraniliprole. In addition, IV d and chlorantraniliprole exhibited comparable potencies against S. frugiperda. Transcriptome analysis showed that the molecular target of compound IV f is the ryanodine receptor. Molecular docking was further performed to verify the mode of action and insecticidal activity against resistant P. xylostella.

Hypothetical protein MAA_07646 is required for stress resistance and pathogenicity in Metarhizium robertsii.

Metarhizium robertsii, a vital entomopathogenic fungus for pest management, relies on various virulence-related proteins for infection. Identifying these proteins, especially those with unknown functions, can illuminate the fungus's virulence mechanisms. Through RNA-seq, we discovered that the hypothetical protein MAA_07646 was significantly upregulated during appressorium formation in M. robertsii. In this study, we characterized MAA_07646, finding its presence in both the nucleus and cytoplasm. Surprisingly, it did not affect vegetative growth, conidiation, or chemical tolerance. However, it played a role in heat and UV radiation sensitivity. Notably, ΔMAA_07646 exhibited reduced virulence in Galleria mellonella larvae due to impaired appressorium formation and decreased expression of virulence-related genes. In conclusion, MAA_07646 contributes to thermotolerance, UV resistance, and virulence in M. robertsii. Understanding its function sheds light on the insecticidal potential of M. robertsii's hypothetical proteins.

Characterization of the serosal cells surrounding Cotesia kariyai larvae and their role in host immunosuppression.

About half of the serosal cells (Scs) of Cotesia kariyai (Ck) eggs are released as teratocytes into the host body cavity after hatching, and another half remain attached to the larval epidermis until the 1st instar larvae of Ck ecdysis to 2nd instars. To investigate the role of the serosal cells surrounding Ck 1st instar larvae (1st Scs) in immune avoidance, Ck 1st instar larvae with and without Scs removed using dispase were transplanted into Mythimna separata larvae (MsLrv), respectively. As a result, Ck 1st instar larvae surrounded by Scs were less susceptible to the MsL encapsulation than Ck 1st instar larvae without the Scs, suggesting that the Scs are involved in suppressing the encapsulation of the MsL. Furthermore, when the granular cells and plasmatocytes of the MsL involved in the encapsulation were incubated in a medium supplemented with proteins extracted from 1st Scs, the plasmatocytes failed to adhere to glass slides, and did not spread their filopodium and lamellipodium. These findings suggest that 1st Scs express proteins that inhibit filopodium and lamellipodium spreading to prevent the MsL plasmatocytes from adhering to Ck larvae.

Opsin mutants alter host plant selection by color vision in the nocturnal invasive pest Tuta absoluta.

In insects, vision is crucial in finding host plants, but its role in nocturnal insects is largely unknown. Vision involves responses to specific spectra of photon wavelengths and opsins plays an important role in this process. Long-wavelength sensitive opsin (LW opsin) and blue-sensitive opsin (BL opsin) are main visual opsin proteins and play important in behavior regulation.We used CRISPR/Cas9 technology to mutate the long-wavelength-sensitive and blue wavelength-sensitive genes and explored the role of vision in the nocturnal invasive pest Tuta absoluta. Light wave experiments revealed that LW2(-/-) and BL(-/-) mutants showed abnormal wavelength tropism. Both LW2 and BL mutations affected the preference of T. absoluta for the green environment. Mutations in LW2 and BL are necessary to inhibit visual attraction. The elimination of LW2 and BL affected the preference of leaf moths for green plants, and mutations in both induced a preference in moths for white plants. Behavioral changes resulting from LW2(-/-) and BL(-/-) mutants were not affected by sense of smell, further supporting the regulatory role of vision in insect behavior. To the best of our knowledge, this is the first study to reveal that vision, not smell, plays an important role in the host-seeking behavior of nocturnal insects at night, of which LW2 and BL opsins are key regulatory factors. These study findings will drive the development of the "vision-ecology" theory.

Key role of the ryanodine receptor I4790K mutation in mediating diamide resistance in Plutella xylostella.

The diamondback moth Plutella xylostella, a global insect pest of cruciferous vegetables, has evolved resistance to many classes of insecticides including diamides. Three point mutations (I4790M, I4790K, and G4946E) in the ryanodine receptor of P. xylostella (PxRyR) have been identified to associate with varying levels of resistance. In this study, we generated a knockin strain (I4790K-KI) of P. xylostella, using CRISPR/Cas9 to introduce the I4790K mutation into PxRyR of the susceptible IPP-S strain. Compared to IPP-S, the edited I4790K-KI strain exhibited high levels of resistance to both anthranilic diamides (chlorantraniliprole 1857-fold, cyantraniliprole 1433-fold) and the phthalic acid diamide flubendiamide (>2272-fold). Resistance to chlorantraniliprole in the I4790K-KI strain was inherited in an autosomal and recessive mode, and genetically linked with the I4790K knockin mutation. Computational modeling suggests the I4790K mutation reduces the binding of diamides to PxRyR by disrupting key hydrogen bonding interactions within the binding cavity. The approximate frequencies of the 4790M, 4790K, and 4946E alleles were assessed in ten geographical field populations of P. xylostella collected in China in 2021. The levels of chlorantraniliprole resistance (2.3- to 1444-fold) in these populations were significantly correlated with the frequencies (0.017-0.917) of the 4790K allele, but not with either 4790M (0-0.183) or 4946E (0.017-0.450) alleles. This demonstrates that the PxRyR I4790K mutation is currently the major contributing factor to chlorantraniliprole resistance in P. xylostella field populations within China. Our findings provide in vivo functional evidence for the causality of the I4790K mutation in PxRyR with high levels of diamide resistance in P. xylostella, and suggest that tracking the frequency of the I4790K allele is crucial for optimizing the monitoring and management of diamide resistance in this crop pest.

EsigPBP3 Was the Important Pheromone-Binding Protein to Recognize Male Pheromones and Key Eucalyptus Volatiles.

Pheromone-binding proteins (PBPs) are specific odorant-binding proteins that can specifically recognize insect pheromones. Through transcriptional analysis of the antennae of adult Endoclita signifer, EsigPBP3 was discovered and identified, and EsigPBP3 was found to be highly expressed in the antennae of male moths. Based on the binding characteristics and ability of EsigPBP3, we can find the key ligands and binding site to consider as a target to control the key wood bore E. signifier. In this study, the fluorescence competitive binding assays (FCBA) showed that EsigPBP3 had a high binding affinity for seven key eucalyptus volatiles. Molecular docking analysis revealed that EsigPBP3 had the strongest binding affinity for the sexual pheromone component, (3E,7E)-4,7,11-trimethyl-1,3,7,10-dodecatetraene. Furthermore, same as the result of FCBA, the EsigPBP3 exhibited high binding affinities to key eucalyptus volatiles, eucalyptol, α-terpinene, (E)-beta-ocimene, (-)-ß-pinene, and (-)-α-pinene, and PHE35, MET7, VAL10, PHE38, ILE52, and PHE118 are key sites. In summary, EsigPBP3 exhibits high binding affinity to male pheromones and key volatile compounds and the crucial binding sites PHE35, MET7, VAL10, PHE38, ILE52, and PHE118 can act as targets in the recognition of E. signifier pheromones.

CncC-Keap1-P450s pathway is involved in the detoxification of emamectin benzoate in the spongy moth Lymantria dispar.

The detoxification of insecticides in insects is dependent on the expression and activity of multiple detoxification enzymes. As an important modulator of detoxification enzymes, the CncC-Keap1 pathway was involved in the detoxification of various pesticides. However, whether the CncC-Keap1 pathway is involved in the detoxification of emamectin benzoate (EMB) is unclear. In this study, we cloned the LdCncC and LdKeap1 from spongy moths (Lymantria dispar). Our results showed that EMB exposure induced oxidative stress, and activated the CncC-Keap1 pathway at mRNA and protein levels. Removing ROS by N-acetylcysteine remarkably decreased H2O2 levels and restored the expression of LdCncC and LdKeap1. The silencing LdCncC, not LdKeap1, by dsRNA significantly decreased the cytochrome P450 activities, and increased the sensitivity of larvae to EMB. Besides, the expression of CYP6B7v1, CYP321A7 and CYP4S4v1 were significantly decreased after silencing LdCncC. Notably, the knockdown of CYP6B7v1, CYP321A7 or CYP4S4v1 significantly increased the mortality induced by EMB exposure. Therefore, we proposed that activation of CncC-Keap1 pathway induced by ROS increased the detoxification of EMB in spongy moths by regulating the expression of CYP6B7v1, CYP321A7 and CYP4S4v1. Our study strengthened the understanding of the detoxification of EMB from the perspective of CncC-Keap1-P450s pathway.

Identification of insect cuticular protein genes LCP17 and SgAbd5 from Helicoverpa armigera and evaluation their roles in fenvalerate resistance.

Insect cuticular protein (ICP) plays an important role in insect growth and development. However, research on the role of ICP in insecticide resistance is very limited. In this study, insect cuticular protein genes LCP17 and SgAbd5 were cloned and characterized in Helicoverpa armigera based on previous transcriptome data. The functions of LCP17 and SgAbd5 genes in fenvalerate resistance were assessed by RNA interference (RNAi), and their response to fenvalerate was further detected. The results showed that LCP17 and SgAbd5 were overexpressed in the fenvalerate-resistant strain comparing with a susceptible strain. The open reading frames of LCP17 and SgAbd5 genes were 423 bp and 369 bp, encoding 141 and 123 amino acids, respectively. LCP17 and SgAbd5 genes were highly expressed in the larval stage, but less expressed in the adult and pupal stages. The expression level of LCP17 and SgAbd5 genes increased significantly after fenvalerate treatment at 24 h. When the cotton bollworms larvae were exposed to fenvalerate at LD50 level, RNAi-mediated silencing of LCP17 and SgAbd5 genes increased the mortality from 50.68% to 68.67% and 63.89%, respectively; the mortality increased to even higher level, which was 73.61%, when these two genes were co-silenced. Moreover, silencing of these two genes caused the cuticle lamellar structure to become loose, which led to increased penetration of fenvalerate into the larvae. The results suggested that LCP17 and SgAbd5 may be involved in the resistance of cotton bollworm to fenvalerate, and LCP17 and SgAbd5 could serve as potential targets for H. armigera control.

Identifying the Key Role of Plutella xylostella General Odorant Binding Protein 2 in Perceiving a Larval Attractant, (E,E)-2,6-Farnesol.

There is currently a lack of effective olfaction-based techniques to control diamondback moth (DBM) larvae. Identifying behaviorally active odorants for DBM larvae and exploring their recognition mechanisms can provide insights into olfaction-based larval control strategies. Through the two-choice assay, (E,E)-2,6-farnesol (farnesol) was identified as a compound exhibiting significant attractant activity toward DBM larvae, achieving an attraction index of 0.48 ± 0.13. PxylGOBP1 and PxylGOBP2, highly expressed in the antennae of DBM larvae, both showed high affinity toward farnesol. RNAi technology was used to knock down PxylGOBP1 and PxylGOBP2, revealing that the attraction of DBM larvae to farnesol nearly vanished following the knockdown of PxylGOBP2, indicating its critical role in recognizing farnesol. Further investigation into the PxylGOBP2-farnesol interaction revealed the importance of residues like Thr9, Trp37, and Phe118 in PxylGOBP2's binding to farnesol. This research is significant for unveiling the olfactory mechanisms of DBM larvae and developing larval behavior regulation techniques.

SfMBP: A novel microbial binding protein and pattern recognition receptor in the fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae).

The fall armyworm, Spodoptera frugiperda, poses a significant threat as a highly destructive agricultural pest in many countries. Understanding the complex interplay between the insect immune system and entomopathogens is critical for optimizing biopesticide efficacy. In this study, we identified a novel microbial binding protein, SfMBP, in S. frugiperda. However, the specific role of SfMBP in the immune response of S. frugiperda remains elusive. Encoded by the LOC118269163 gene, SfMBP shows significant induction in S. frugiperda larvae infected with the entomopathogen Beauveria bassiana. Consisting of 115 amino acids with a signal peptide, an N-terminal flexible region and a C-terminal ß-sheet, SfMBP lacks any known functional domains. It is expressed predominantly during early larval stages and in the larval epidermis. Notably, SfMBP is significantly induced in larvae infected with bacteria and fungi and in SF9 cells stimulated by peptidoglycan. While recombinant SfMBP (rSfMBP) does not inhibit bacterial growth, it demonstrates binding capabilities to bacteria, fungal spores, peptidoglycan, lipopolysaccharides, and polysaccharides. This binding is inhibited by monosaccharides and EDTA. Molecular docking reveals potential Zn2+-interacting residues and three cavities. Furthermore, rSfMBP induces bacterial agglutination in the presence of Zn2+. It also binds to insect hemocytes and SF9 cells, enhancing phagocytosis and agglutination responses. Injection of rSfMBP increased the survival of S. frugiperda larvae infected with B. bassiana, whereas blocking SfMBP with the antibody decreased survival. These results suggest that SfMBP acts as a pattern recognition receptor that enhances pathogen recognition and cellular immune responses. Consequently, this study provides valuable insights for the development of pest control measures.

Molecular and Functional Characterization of Three General Odorant-Binding Protein 2 Genes in Cydia pomonella (Lepidoptera: Tortricidae).

General odorant-binding proteins (GOBPs) play a crucial role in the detection of host plant volatiles and pheromones by lepidopterans. Previous studies identified two duplications in the GOBP2 gene in Cydia pomonella. In this study, we employed qRT-PCR, protein purification, and fluorescence competitive binding assays to investigate the functions of three GOBP2 genes in C. pomonella. Our findings reveal that CpomGOBP2a and CpomGOBP2b are specifically highly expressed in antennae, while CpomGOBP2c exhibits high specific expression in wings, suggesting a potential divergence in their functions. Recombinant proteins of CpomGOBP2a, CpomGOBP2b, and CpomGOBP2c were successfully expressed and purified, enabling an in-depth exploration of their functions. Competitive binding assays with 20 host plant volatiles and the sex pheromone (codlemone) demonstrated that CpomGOBP2a exhibits strong binding to four compounds, namely butyl octanoate, ethyl (2E,4Z)-deca-2,4-dienoate (pear ester), codlemone, and geranylacetone, with corresponding dissolution constants (Ki) of 8.59993 µM, 9.14704 µM, 22.66298 µM, and 22.86923 µM, respectively. CpomGOBP2b showed specific binding to pear ester (Ki = 17.37481 µM), while CpomGOBP2c did not exhibit binding to any tested compounds. In conclusion, our results indicate a functional divergence among CpomGOBP2a, CpomGOBP2b, and CpomGOBP2c. These findings contribute valuable insights for the development of novel prevention and control technologies and enhance our understanding of the evolutionary mechanisms of olfactory genes in C. pomonella.

Stored alcohol and fatty acid intermediates and the biosynthesis of sex pheromone aldehyde in the moth Chloridea virescens.

In most species of moths, the female produces and releases a volatile sex pheromone from a specific gland to attract a mate. Biosynthesis of the most common type of moth sex pheromone component (Type 1) involves de novo synthesis of hexadecanoate (16:Acyl), followed by modification to various fatty acyl intermediates, then reduction to a primary alcohol, which may be acetylated or oxidized to produce an acetate ester or aldehyde, respectively. Our previous work on the moth Chloridea virescens (Noctuidae) showed that females produce 90% of the major pheromone component, (Z)-11-hexadecenal (Z11-16:Ald), via a direct and rapid route of de novo biosynthesis with highly labile intermediates, and ca. 10% from an indirect route that likely mobilizes a pre-synthesized 16-carbon skeleton, possibly, (Z)-11-hexadecenoate (Z11-16:Acyl) or hexadecanoate (16:Acyl). In this paper, we use stable isotope tracer/tracee techniques to study the dynamics of the precursor alcohol (Z)-11-hexadecenol (Z11-16:OH) and stores of Z11-16:Acyl and 16:Acyl to determine their roles in biosynthesis of Z11-16:Ald. We found: (i) that intracellular Z11-16:OH is synthesized at roughly the same rate as Z11-16:Ald, indicating that translocation and oxidation of this moiety does not rate limit biosynthesis of Z11-16:Ald, (ii) intracellular Z11-16:OH consists of two pools, a highly labile one rapidly translocated out of the cell and converted to Z11-16:Ald, and a less labile one that mostly remains in gland cells, (iii) during pheromone biosynthesis, net stores of Z11-16:Acyl increase, suggesting it is not the source of Z11-16:Ald produced by the indirect route, and (iv) no evidence for the gland synthesizing stored 16:Acyl prior to (up to 2 days before eclosion), or after, synthesis of pheromone commenced, suggesting the bulk of this stored moiety is synthesized elsewhere and transported to the gland prior to gland maturation. Thus, the pheromone gland of C. virescens produces very little stored fat over its functional lifetime, being optimized to produce sex pheromone.

Involvement of miR-8510a-3p in response to Cry1Ac protoxin by regulating PxABCG3 in Plutella xylostella.

Overuse of insecticides has accelerated the evolution of insecticide resistance and created serious environmental concerns worldwide, thus incentivizing development of alternative methods. Bacillus thuringiensis (Bt) is an insecticidal bacterium that has been developed as a biopesticide to successfully control multiple species of pests. It operates by secreting several insect toxins such as Cry1Ac. However, metabolic resistance based on ATP-binding cassette (ABC) transporters may play a crucial role in the development of metabolic resistance to Bt. Here, we characterized an ABCG gene from the agricultural pest Plutella xylostella (PxABCG3) and found that it was highly expressed in a Cry1Ac-resistant strain, up-regulated after Cry1Ac protoxin treatment. Binding miR-8510a-3p to the coding sequence (CDS) of PxABCG3 was then confirmed by a luciferase reporter assay and RNA immunoprecipitation. miR-8510a-3p agomir delivery markedly reduced PxABCG3 expression in vivo and consequently decreased the tolerance of P. xylostella to Cry1Ac, while reduction of miR-8510a-3p significantly increased PxABCG3 expression, accompanied by an increased tolerance to Cry1Ac. Our results suggest that miR-8510a-3p could potentially be used as a novel molecular target against P. xylostella or other lepidopterans, providing novel insights into developing effective and environmentally friendly pesticides.