Single Electron Transfer Reductive Deuteration of Acyl Chlorides for the Synthesis of Deuterated Alcohols with a High Deuterium Atom Economy.

We present a highly deuterium atom economical approach for the synthesis of deuterated alcohols via the single electron transfer (SET) reductive deuteration of acyl chlorides. Cost-effective sodium dispersion and EtOD-d1 were used as the single electron donor and deuterium donor, respectively. Our approach achieved up to 49% deuterium atom economy, which represents the highest deuterium atom economy yet achieved in SET reductive deuteration reactions. With all 20 tested substrates, excellent regioselectivity and >92% deuterium incorporations were obtained. Furthermore, we demonstrated the potential of this methodology by synthesizing four deuterated analogues of pesticides.

The knockout of the nicotinic acetylcholine receptor subunit gene α1 (nAChR α1) through CRISPR/Cas9 technology exposes its involvement in the resistance of Spodoptera exigua to insecticides.

Insect nicotinic acetylcholine receptors (nAChRs) are the directed targets of many insecticides. However, there have been no reports on the molecular characterization of the nAChR gene family or the causal association between nAChR α1 and resistance to insecticides in S. exigua, which is a significant agricultural pest. In this study, we identified a total of 9 candidate nAChR subunits in S. exigua, namely nAChR α1-α7 and nAChR ß1-ß2. For functional validation roles of Seα1 in insecticide resistance of S. exigua, we introduced a âˆ¼ 1041-bp deletion of the Seα1 gene in a homozygous mutant strain (Seα1-KO) by CRISPR/Cas9 genome editing system, resulting in a premature truncation of the Seα1 protein and the subsequent loss of functional transmembrane (TM) 3 and TM4 elements. Compared with WH-S strain (wild-type strain), the Seα1-KO strain exhibited 2.62-folds resistant to trifluoropyrimidine, 8.3-folds resistant to dimehypo, and 5.28-folds resistant to dinotefuran, but no significant change in susceptibility to emamectin benzoate, spinetoram, lambda-cyhalothrin, permethrin and chlorpyrifos. Thus, this study has laid a solid foundation for investigating the role of nAChRs in S. exigua, and provides evidence for the crucial involvement of the α1 subunit in the mechanism of trifluoropyrimidine, dimehypo, and dinotefuran in S. exigua. Moreover, it provides a reference for the value of Seα1 subunit and its homologues in other species as insecticide targets.

Periplocoside P affects synaptic transmission at the neuromuscular junction and reduces synaptic excitability in Drosophila melanogaster by inhibiting V-ATPase.

BACKGROUND: Periplocoside P (PSP) is a major component of Periploca sepium Bunge known for its potent insecticidal activity. V-Type adenosine triphosphatase (V-ATPase), which is widely distributed in the cytoplasmic membranes and organelles of eukaryotic cells, plays a crucial role in synaptic excitability conduction. Previous research has shown that PSP targets the apical membrane of goblet cells in the insect midgut. However, the effects of PSP on synaptic transmission at the neuromuscular junction are often overlooked. RESULTS: The bioassay revealed that Drosophila adults with different genetic backgrounds showed varying levels of susceptibility to PSP in the order: parats1 > parats1 ;DSC1-/- ≈ w1118 > DSC1-/- . Intracellular electrode recording demonstrated that PSP, similar to bafilomycin A1, had an impact on the amplitude of the excitatory junction potential (EJP) and accelerated excitability decay. Furthermore, the alteration in EJP amplitude is concentration-dependent. Another surprising discovery was that the knockout DSC1 channel showed insensitivity to PSP. CONCLUSION: Our findings confirm that PSP can influence synaptic transmission at the neuromuscular junction of Drosophila larvae by targeting V-ATPase. These results provide a basis for investigating the mechanism of action of PSP and its potential application in designing novel insecticides. © 2023 Society of Chemical Industry.

Identification and functional characterization of the dirigent gene family in Phryma leptostachya and the contribution of PlDIR1 in lignan biosynthesis.

BACKGROUND: Furofuran lignans, the main insecticidal ingredient in Phryma leptostachya, exhibit excellent controlling efficacy against a variety of pests. During the biosynthesis of furofuran lignans, Dirigent proteins (DIRs) are thought to be dominant in the stereoselective coupling of coniferyl alcohol to form ( ±)-pinoresinol. There are DIR family members in almost every vascular plant, but members of DIRs in P. leptostachya are unknown. To identify the PlDIR genes and elucidate their functions in lignan biosynthesis, this study performed transcriptome-wide analysis and characterized the catalytic activity of the PlDIR1 protein. RESULTS: Fifteen full-length unique PlDIR genes were identified in P. leptostachya. A phylogenetic analysis of the PlDIRs classified them into four subfamilies (DIR-a, DIR-b/d, DIR-e, and DIR-g), and 12 conserved motifs were found among them. In tissue-specific expression analysis, except for PlDIR7, which displayed the highest transcript abundance in seeds, the other PlDIRs showed preferential expression in roots, leaves, and stems. Furthermore, the treatments with signaling molecules demonstrated that PlDIRs could be significantly induced by methyl jasmonate (MeJA), salicylic acid (SA), and ethylene (ETH), both in the roots and leaves of P. leptostachya. In examining the tertiary structure of the protein and the critical amino acids, it was found that PlDIR1, one of the DIR-a subfamily members, might be involved in the region- and stereo-selectivity of the phenoxy radical. Accordingly, LC-MS/MS analysis demonstrated the catalytic activity of recombinant PlDIR1 protein from Escherichia coli to direct coniferyl alcohol coupling into ( +)-pinoresinol. The active sites and hydrogen bonds of the interaction between PlDIR1 and bis-quinone methide (bisQM), the intermediate in ( +)-pinoresinol formation, were analyzed by molecular docking. As a result, 18 active sites and 4 hydrogen bonds (Asp-42, Ala-113, Leu-138, Arg-143) were discovered in the PlDIR1-bisQM complex. Moreover, correlation analysis indicated that the expression profile of PlDIR1 was closely connected with lignan accumulations after SA treatment. CONCLUSIONS: The results of this study will provide useful clues for uncovering P. leptostachya's lignan biosynthesis pathway as well as facilitate further studies on the DIR family.

Effects of periplocoside T isolated from Periploca sepium on behavior and sensory-CNS-motor circuits in Drosophila melanogaster larvae.

Periplocoside T (PST) from Periploca sepium has insecticidal activity against some lepidopterans, which can significantly inhibit the activity of vacuolar-type H+-ATPases (V-ATPase). V-ATPase is involved in the release of neurotransmitters in vesicles during nerve signal transduction. However, there are actions of PST on behavior and sensory-central nervous system (CNS)-motor neural circuit which are commonly overlooked. After exposure to 500 mg/L PST for 48 h, the difference of the proportion of larvae responding to stimuli in the four Drosophila strains was not significant as compared to controls, but larval mouth hook movement and body wall motion were significantly decreased as compared to controls, and the decrease was more obvious in parats1; DSC1-/- and DSC1-/- strains, especially in parats1; DSC1-/- strain. Compared with control (DMSO), the excitatory junction potential (EJP) frequencies of sensory-CNS-motor circuits in the four Drosophila strains after PST or bafiloymcin A1 (BA1, a V-ATPase specific inhibitor) treatment gradually decreased with time, and the decreasing amplitude of BA1 treatment was greater than that of PST treatment, but both were higher than that of the control. The decay amplitude of EJP frequency in two strains with DSC1 channel knockout was lower than that of w1118 and parats1 strains without DSC1 channel knockout. Thus, the results indicated that PST, similar to BA1, could inhibit the transmission of sensory-CNS-motor circuit excitability of Drosophila larvae by inhibiting the activity of V-ATPase, and DSC1 channel play a role of in regulating the stability of nervous system.

Role of DSC1 in Drosophila melanogaster synaptic activities in response to haedoxan A.

Drosophila sodium channel 1 (DSC1) encodes a voltage-gated divalent cation channel that mediates neuronal excitability in insects. Previous research revealed that DSC1 knockout Drosophila melanogaster conferred different susceptibility to insecticides, which indicated the vital regulation role of DSC1 under insecticide stress. Haedoxan A (HA) is a lignan compound isolated from Phryma leptostachya, and we found that HA has excellent insecticidal activity and is worthy of further study as a botanical insecticide. Herein, we performed bioassay and electrophysiological experiments to test the biological and neural changes in the larval Drosophila with/without DSC1 knockout in response to HA. Bioassay results showed that knockout of DSC1 reduced the sensitivity to HA in both w1118 (a common wild-type strain in the laboratory) and parats1 (a pyrethroid-resistant strain) larvae. Except for parats1 /DSC1-/- , electrophysiology results implicated that HA delayed the decay rate and increased the frequency of miniature excitatory junctional potentials of Drosophila from w1118 , parats1 , and DSC1-/- strains. Moreover, the neuromuscular synapse excitatory activities of parats1 /DSC1-/- larvae were more sensitive to HA than DSC1-/- larvae, which further confirmed the functional contribution of DSC1 to neuronal excitability. Collectively, these results indicated that the DSC1 channel not only regulated the insecticidal activity of HA, but also maintained the stability of neural circuits through functional interaction with voltage-gated sodium channels. Therefore, our study provides useful information for elucidating the regulatory mechanism of DSC1 in the neural system of insects involving the action of HA derived from P. leptostachya.

Design, synthesis and insecticidal activities of 4-propargyloxybenzene sulfonamide derivatives substituted with amino acids.

Sixty-nine 4-propargyloxybenzene sulfonamide derivatives with different amino acids as amino substituent were synthesized and evaluated for their insecticidal activity against third-instar Mythimna separate. The bioassay results revealed that some derivatives bearing amino acid ester group performed good insecticidal activity against third-instar M.separata, such as the LC50 values of D18 and D19 were 4.28 and 2.96 mg/ml after 48 h, in particular, the LC50 of D16 was 2.38 mg/ml and the activity was improved by 14 times compared to celangulin V (34.48 mg/ml). The above results provided theoretical and experimental basis for the discovery of novel insecticidal active compounds.

Functional validation of CYP304A1 associated with haedoxan A detoxification in Aedes albopictus by RNAi and transgenic drosophila.

BACKGROUND: Insect cytochrome P450 monooxygenases play important roles in the detoxification metabolism of endogenous and exogenous compounds. Haedoxan A (HA) from Phryma leptostachya L. is a highly efficient natural pesticide used to control houseflies and mosquitos. CYP4C21 and CYP304A1 were previously demonstrated to be transcriptionally increased in Aedes albopictus in response to HA exposure, but their involvement in HA metabolism is unknown. RESULTS: Our data showed that CYP304A1 expression levels in A. albopictus were highest in third-instar larvae, and the expression level of CYP4C21 decreased significantly with the growth of instars, with the lowest occurring in the pupal stage. Compared with the control, the silencing of CYP304A1 and CYP4C21 genes by chitosan nanoparticle-mediated RNA interference could deplete 58.2% and 54.0% of the expression of corresponding genes, respectively. The bioassay data showed that knocking down the expression of CYP304A1 increased the mortality of A. albopictus when exposed to HA at LC30 and LC50 doses, but did not significantly increase mortality after silencing CYP4C21. Our data demonstrated that CYP304A1, but not CYP4C21, may be involved in HA detoxification. Moreover, the resistance ratio of CYP304A1 overexpressing flies was approximately 2-fold higher than that of the control line. The metabolized product of HA by CYP304A1 needs to be further confirmed by in vitro expression. CONCLUSION: This finding showed that inducibility was not always linked to detoxifying capabilities, and enhanced our understanding of the molecular basis of HA metabolic detoxification in A. albopictus. © 2022 Society of Chemical Industry.

A Genetic Compensation Phenomenon and Global Gene Expression Changes in Sex-miR-2766-3p Knockout Strain of Spodoptera exigua Hübner (Lepidoptera: Noctuidae).

MicroRNAs (miRNAs) drive the post-transcriptional repression of target mRNAs and play important roles in a variety of biological processes. miR-2766-3p is conserved and abundant in Lepidopteran species and may be involved in a variety of biological activities. In this study, Sex-miR-2766-3p was predicted to potentially bind to the 3' untranslated region (UTR) of cap 'n' collar isoform C (CncC) in Spodoptera exigua, and Sex-miR-2766-3p was confirmed to regulate the expression of SeCncC through screening with a luciferase reporter system. Although CRISPR/Cas9 has been extensively utilized to examine insect gene function, studies of miRNA function are still relatively uncommon. Thus, we employed CRISPR/Cas9 to knock out Sex-miR-2766-3p from S. exigua. However, the expression of SeCncC was not significantly altered in the knockout strain (2766-KO) compared with that of the WHS strain. This result suggested that a miRNA knockout might lack phenotypes because of genetic robustness. Additionally, we used transcriptome analysis to examine how the global gene expression patterns of the Sex-miR-2766-3p knockout strain varied. RNA-seq data revealed 1746 upregulated and 2183 downregulated differentially expressed genes (DEGs) in the 2766-KO strain, which might be the result of Sex-miR-2766-3p loss or DNA lesions as the trigger for transcriptional adaptation. GO function classification and KEGG pathway analyses showed that these DEGs were enriched for terms related to binding, catalytic activity, metabolic process, and signal transduction. Our findings demonstrated that S. exigua could compensate for the missing Sex-miR-2766-3p by maintaining the expression of SeCncC by other pathways.

Phylogeny and functional characterization of the cinnamyl alcohol dehydrogenase gene family in Phryma leptostachya.

Phryma leptostachya has attracted increasing attention because it is rich in furofuran lignans with a wide range of biological activities. Biosynthesis of furofuran lignans begins with the dimerization of coniferyl alcohol, one of the monolignol. Cinnamyl alcohol dehydrogenase (CAD) catalyzes the final step of monolignol biosynthesis, reducing cinnamyl aldehydes to cinnamyl alcohol. As it is in the terminal position of monolignol biosynthesis, its type and activity can cause significant changes in the total amount and composition of lignans. Herein, combined with bioinformatics analysis and in vitro enzyme assays, we clarified that CAD in P. leptostachya belonged to a multigene family, and identified nearly the entire CAD gene family. Our in-depth characterization about the functions and structures of two major CAD isoforms, PlCAD2 and PlCAD3, showed that PlCAD2 exhibited the highest catalytic activity, and coniferyl aldehyde was its preferred substrate, followed by PlCAD3, and sinapyl aldehyde was its preferred substrate. Considering the accumulation patterns of furofuran lignans and expression patterns of PlCADs, we speculated that PlCAD2 was the predominant CAD isoform responsible for furofuran lignans biosynthesis in P. leptostachya. Moreover, these CADs found here can also provide effective biological parts for lignans and lignins biosynthesis.

Evidence for Multiple Origins of Knockdown Resistance (kdr) in Spodoptera exigua (Hübna) (Lepidoptera: Noctuidae) From China.

The beet armyworm, Spodoptera exigua (Hübna) is a serious agricultural pest that is challenging to control due to resistance to most pesticides, including pyrethroids. This resistance has previously been linked to the knockdown resistance (kdr) mutation (L1014F) of the voltage-gated sodium channel (VGSC) in S. exigua. To better understand the frequencies of the kdr mutation of SeVGSC and identify the evolutionary origins of kdr mutation in S. exigua, seven populations of S. exigua were collected in China, and partial SeVGSC genomic sequences for each individual were acquired. The bioassays showed that the survival rates of seven populations of S. exigua larvae exposed to the discriminating dose of beta-cypermethrin (0.05 mg/cm2) ranged from 91.66% to 100%, indicating that all seven populations had evolved resistance to beta-cypermethrin. The frequencies of kdr mutation (CTT to TTT) of SeVGSC of field populations ranged China were from 60% to 89.6%. The CTT to CAT substitution at this coding position resulting in the L1014H (kdr-H) mutation was found in only one individual from the QP18 population. Based on the phylogeny of SeVGSC alleles, it appeared that the kdr mutation in S. exigua populations had multiple origins, which has major consequences for pyrethroid effectiveness in the field. Thus, it is recommended to limit the use of pyrethroid and encourage rotation of insecticides with different modes of action for control of S. exigua to alleviate resistance development.

Knockin of the G275E mutation of the nicotinic acetylcholine receptor (nAChR) α6 confers high levels of resistance to spinosyns in Spodoptera exigua.

Spinosyns, including spinosad and spinetoram, act on the insect central nervous system, gradually paralyzing or destroying the target insect. Spinosad resistance is associated with loss-of-function mutations in the nicotinic acetylcholine receptor (nAChR) α6 subunit in a number of agricultural pests. Using gene editing, nAChR α6 has been verified as a target for spinosyns in five insect species. Recently, a point mutation (G275E) in exon 9 of nAChR α6 was identified in spinosad-resistant strains of Thrips palmi and Tuta absoluta. To date, no in vivo functional evidence has been obtained to support that this mutation is involved in spinosyn resistance in lepidopteran pests. In this study, the G275E mutation was introduced into the nAChR of Spodoptera exigua using clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated protein 9 (Cas9) gene-editing technology. Reverse transcriptase-polymerase chain reaction and sequencing confirmed that this mutation was present in exon 9 of the nAChR transcripts in the edited 275E strain. The results of bioassays showed that the 275E strain was highly resistant to spinosad (230-fold) and spinetoram (792-fold) compared to the unedited background strain, directly confirming that the G275E mutation of the nAChR α6 subunit confers high levels of spinosyn resistance in S. exigua. Inheritance analysis showed that the resistance trait is autosomal and incompletely recessive. This study employs a reverse genetics approach to validate the functional role played by the G275E mutation in nAChR α6 of S. exigua in spinosyns resistance and provides another example of the use of CRISPR/Cas9 gene-editing technology to confirm the role played by candidate target site mutations in insecticide resistance.

Negative cross-resistance of a pyrethroid-resistant Drosophila mutant to Phryma leptostachya-derived haedoxan A.

Voltage-gated sodium channels are the primary target of pyrethroid insecticides. Mutations in sodium channel confer knockdown resistance (kdr) to pyrethroids in various arthropod pests. Haedoxan A (HA) is the major insecticidal component from Phryma leptostachya. It has been shown that HA alters electrical responses at the Drosophila neuromuscular junction and modifies the gating properties of cockroach sodium channels expressed in Xenopus oocytes. However, whether sodium channel mutations that confer pyrethroid resistance also affect the action of HA is unknown. In this study, we conducted bioassays using HA and permethrin in two Drosophila melanogaster strains: w1118 , an insecticide-susceptible strain, and parats1 , a pyrethroid-resistant strain due to a I265N mutation in the sodium channel, and identified a new case of negative cross-resistance (NCR) between permethrin and HA. Both parats1 larvae and adults were more resistant to permethrin, as expected. However, both parats1 larvae and adults were more sensitive to HA compared to w1118 . We confirmed that the I265N mutation reduced the sensitivity to permethrin of a Drosophila sodium channel variant, DmNav 22, expressed in Xenopus oocytes. Interestingly, the I265N mutation also abolished the effect of HA on sodium channels. Further characterization showed that I265 on the sodium channels is critical for the action of both pyrethroids and HA on sodium channels, pointing to an overlapping mode of action between pyrethroids and HA on the sodium channel. Overall, our results suggest an I265N-independnt mechanism(s) in parats1 flies that is responsible for the NCR between permethrin and HA at the whole insect level.

Transcriptome Analysis of Aedes albopictus (Diptera: Culicidae) Larvae Exposed With a Sublethal Dose of Haedoxan A.

Aedes albopictus is the vector of arbovirus diseases including yellow fever, dengue, Zika virus, and chikungunya fever, and it poses an enormous threat to human health worldwide. Previous studies have revealed that haedoxan A (HA), which is an insecticidal sesquilignan from Phryma leptostachya L., is a highly effective natural insecticide for managing mosquitoes and houseflies; however, the mechanisms underlying the response of Ae. albopictus after treatment with sublethal concentrations of HA is not clear. Here, high-throughput sequencing was used to analyze the gene expression changes in Ae. albopictus larvae after treatment with the LC30 of HA. In total, 416 differentially expressed genes (DEGs) were identified, including 328 upregulated genes and 88 downregulated genes. Identification and verification of related DEGs were performed by RT-qPCR. The results showed that two P450 unigenes (CYP4C21 and CYP304A1), one carboxylesterase, and one ABC transporter (ABCG1) were induced by HA, which indicated that these detoxifying enzyme genes might play a major role in the metabolic and detoxification processes of HA. Additionally, acetylcholine receptor subunit ɑ2 (AChRα2), AChRα5, AChRα9, and the glutamate receptor ionotropic kainate 2 (GRIK2) were found to be upregulated in HA-treated larvae, suggesting that HA affected the conduction of action potentials and synaptic transmission by disrupting the function of neural receptors. These results provide a foundation for further elucidating the target of HA and the mechanism of detoxification metabolism in Ae. albopictus.

Biochemical and phytoremediation of Plantago major L. to protect tomato plants from the contamination of cypermethrin pesticide.

Phytoremediation is an environmentally friendly therapy to minimize soil pollution. Cypermethrin (CYP) is one of the most frequently used pyrethroid insecticides against a variety of pests. We aimed at evaluating the potential of using an economic plant like tomato (Solanum lycopersicum L.) as a control alone and together with Plantago major L. (PM) for the uptake of CYP residue from contaminated soil, also, investigating the antioxidant enzymes such as (SOD, POD, and CAT) in roots of PM and tomato. For the first time, we studied the intercropping between PM on tomato plants for the uptake of CYP residue from contaminated soil and phytoremediation of PM as a curative plant to save tomato plants from CYP residue. In a pot experiment, we have cultivated PM and tomato in soil polluted with CYP (10 µg g-1). Data showed that PM and tomato accumulated significant amounts of CYP in their tissues. However, PM is better than tomato in uptake CYP from the soil. The longest half-life value (t1/2) of CYP was in PM + tomato together treatment (12.7 days), and the shortest was in the soil with tomato alone (6.81 days). Moreover, the activity of SOD, POD, and CAT in treated tomato and PM roots significantly (p > 0.05) exceeded control plants after 8 days from exposure. In this study, a good strategy was recommended to uptake CYP residue from soil by PM and protect tomato plants from CYP residue as well as safe for human and non-target organisms.

Identification and mechanism of insecticidal periplocosides from the root bark of Periploca sepium Bunge.

BACKGROUND: The Periploca sepium bark root (PSBR) has been regarded as a potential botanical insecticide because of its significant insecticidal activity of secondary metabolites. Several periplocosides were isolated from it as promising pesticides to control crop pests in agriculture. RESULTS: In our research, two new periplocosides, along with four known periplocosides were isolated from PSBR. The names of new periplocosides were periplocoside T (PST) and periplocoside U (PSU) while another four periplocosides were known as follows: periplocoside A (PSA), periplocoside F (PSF), periplocoside E (PSE) and periplocoside D (PSD). All periplocosides were evalulated for insecticidal activity against 3rd Mythimna separata (Walker) and Plutella xylostella. The biometric data showed that periplocoside T, PSD and PSF had remarkable insecticidal activity against tested insects. Its values of LD50 were 1.31, 3.94 and 3.42 µg·lavare-1 against 3rd M. separata respectively, while the activity of those compounds against 3rd P. xylostella were 5.45, 12.17 and 13.95 µg·lavare-1 , respectively. It was apparent after further study of the mechanism of action against M. separata was conducted that PST possessed the most significant insecticidal activity. The results of enzymatic activity displayed that powerful activation of tryptase, especially weak alkaline tryptase might be a dominant factor causing death of M. separata in vivo. CONCLUSION: We herein report isolation and the mechanisms of action of insecticidal periplocosides, which established the fundamental development of natural agents to prevent pest damage to crops. © 2020 Society of Chemical Industry.

Transcriptome analysis of Plantago major as a phytoremediator to identify some genes related to cypermethrin detoxification.

Cypermethrin (CYP) is a toxic manmade chemical compound belonging to pyrethroid insecticides contaminating the environment. Plantago major (PM) has numerous excellent advantages like high biomass yield and great stress tolerance, which make it able to increase the efficacy of phytoremediation. So far, no study has directly or indirectly made a transcriptome analysis (RNA-seq) of PM under CYP stress. The aim of this study is to identify the genes in PM related to CYP detoxification (10 µg mL-1) and compared with control. In this study, BGISEQ-500 high-throughput sequencing technology independently developed by BGI was used to sequence the transcriptome of P. major. Six libraries were constructed including (CK_1, CK_2, and CK_3) and (CYP_1, CYP_2, and CYP_3) were sequenced for transcripts involved in CYP detoxification. Our data showed that de novo assembly generated 138,806 unigenes with an average length of 1129 bp. Analyzing the annotation results of the KEGG database between the samples revealed 37,177 differentially expressed genes (DEGs), 18,062 down- and 19,115 upregulated under CYP treatment compared with control. A set of 107 genes of cytochrome P450 (Cyt P450), 43 genes of glutathione S-transferases (GST), 25 genes of glycosyltransferases (GTs), 113 genes from ABC transporters, 21 genes from multidrug and toxin efflux (MATE), 11 genes from oligopeptide transporter (OPT), and 3 genes of metallothioneins (MT) were upregulated notably. By using quantitative real-time PCR (qRT-PCR), the results of gene expression for 12 randomly selected DEGs were confirmed, showing the different patterns of response to CYP in PM tissues. Furthermore, the enzyme activity of Cyt P450 and GST in PM under CYP stress was significantly increased in roots and leaves than in control. This study introduces a clue to understand the metabolic pathways of plants used in phytoremediation by identifying the highly expressed genes related to phytoremediation which would be utilized to enhance pesticide detoxification and reduce pollution problem.

Functional validation of nicotinic acetylcholine receptor (nAChR) α6 as a target of spinosyns in Spodoptera exigua utilizing the CRISPR/Cas9 system.

BACKGROUND: The beet armyworm, Spodoptera exigua, is a serious agricultural pest that is primarily controlled using chemical insecticides. Recently, resistance to the insecticide spinosad has been described in S. exigua field populations. To date, there has been no functional evidence proving the involvement of the nicotinic acetylcholine receptor (nAChR) α6 mutation in spinosad resistance in S. exigua. RESULTS: In this study, using the CRISPR/Cas9 genome-editing system, a homozygous strain (Seα6-KO) with approximately 1760-bp deletion within Seα6 in S. exigua causing a premature truncation of Seα6 was successfully constructed. Insecticide bioassays showed that Seα6-KO exhibited 373-fold higher resistance to spinosad and 850-fold higher resistance to spinetoram compared to WH-S strain with the same genetic background but showed no significant change in susceptibility to emamectin benzoate and chlorantraniliprole. Genetic analysis revealed that Seα6-KO is inherited as an incompletely recessive trait. CONCLUSION: The results clearly demonstrated the functional role of Seα6 in resistance to spinosyn insecticides and provide an example of using genome editing to verify a target premature truncation associated with resistance.

Insecticidal Activity of Four Lignans Isolated from Phryma leptostachya.

A new lignan (T4) and three known lignans (T1, T2, and T3) were isolated from the methanol extract of the roots of Phryma leptostachya using bioassay-guided method, and their structures were identified as phrymarolin I (T1), II (T2), haedoxan A (T3), and methyl 4-((6a-acetoxy-4-(6-methoxybenzo[d][1,3]dioxol-5-yl)tetrahydro-1H,3H-furo[3,4-c]furan-1-yl)oxy)-1-hydroxy-2,2-dimethoxy-5-oxocyclopent-3-ene-1-carboxylate (T4) byNMR and ESI-MS spectral data. Bioassay results revealed that haedoxan A exhibited remarkably high insecticidal activity against Mythimna separata with a stomach toxicity LC50 value of 17.06 mg/L and a topical toxicity LC50 value of 1123.14 mg/L at 24 h, respectively. Phrymarolin I and compound T4 also showed some stomach toxicity against M. separata with KD50 values of 3450.21 mg/L at 4 h and 2807.10 mg/L at 8 h, respectively. In addition, phrymarolin I and haedoxan A exhibited some stomach toxicity against Plutella xylostella with an LC50 value of 1432.05 and 857.28 mg/L at 48 h, respectively. In conclusion, this study demonstrated that lignans from P. leptostachya are promising as a novel class of insecticides or insecticide lead compounds for developing botanical pesticides.

De novo leaf and root transcriptome analysis to explore biosynthetic pathway of Celangulin V in Celastrus angulatus maxim.

BACKGROUND: Celastrus angulatus Maxim is a kind of crucial and traditional insecticidal plant widely distributed in the mountains of southwest China. Celangulin V is the efficient insecticidal sesquiterpenoid of C. angulatus and widely used in pest control in China, but the low yield and discontinuous supply impeded its further popularization and application. Fortunately, the development of synthetic biology provided an opportunity for sustainable supply of Celangulin V, for which understanding its biosynthetic pathway is indispensable. RESULTS: In this study, six cDNA libraries were prepared from leaf and root of C. angulatus before global transcriptome analyses using the BGISEQ-500 platform. A total of 104,950 unigenes were finally obtained with an average length of 1200 bp in six transcriptome databases of C. angulatus, in which 51,817 unigenes classified into 25 KOG classifications, 39,866 unigenes categorized into 55 GO functional groups, and 48,810 unigenes assigned to 135 KEGG pathways, 145 of which were putative biosynthetic genes of sesquiterpenoid and triterpenoid. 16 unigenes were speculated to be related to Celangulin V biosynthesis. De novo assembled sequences were verified by Quantitative Real-Time PCR (qRT-PCR) analysis. CONCLUSIONS: This study is the first report on transcriptome analysis of C. angulatus, and 16 unigenes probably involved in the biosynthesis of Celangulin V were finally collected. The transcriptome data will make great contributions to research for this specific insecticidal plant and the further gene mining for biosynthesis of Celangulin V and other sesquiterpene polyol esters.