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Voltage-gated potassium channel Kv2 is the primarily delayed rectifier in insect nerves and muscles involved in several crucial biological processes, including action potential regulation, photoreceptor performance, and larval locomotor. It is a potential molecular target for developing a novel pesticide for mosquitos. However, there are few studies on the Kv2 channel in agricultural pests.The only α-subunit gene of the Kv2 channel in Plutella xylostella (L.), PxShab, was cloned, and its expression profile was analyzed. The relative expression level of PxShab was highest in the pupal stage of both sexes and male adults but lowest in female adults. Meanwhile, PxShab had the highest expression in the head in both larvae and adults. Then, PxShab was stably expressed in the HEK-293 T cell line. Whole cell patch clamp recordings showed an outward current whose current-voltage relationship conformed to a typical delayed-rectifier potassium channel. 20 μM quinidine could effectively inhibit the potassium current, while the channel was insensitive to 4-AP even at 10 mM. Several potential compounds and botanical pesticides were assessed, and carvedilol (ICThis study revealed the pharmacological properties of PxShab and screened out several high potency inhibitors, which laid the foundation for further functional research of PxShab and provides new insight into designing novel insecticides. © 2022 Society of Chemical Industry.

Two novel strains of actinobacteria, ZYC-3T and BH-SS-21T, were isolated from Hunan Province, PR China. The fermentation broth of BH-SS-21T inhibited the rapid spread of ginger blast, unlike that of ZYC-3T. The taxonomic characteristics of ZYC-3T and BH-SS-21T were defined using a polyphasic approach. The analysis of the full-length 16S rRNA gene sequence revealed that ZYC-3T and BH-SS-21T represented members of the genus Streptomyces . ZYC-3T had less than 98.7% sequence similarities to all species of the genus Streptomyces , while BH-SS-21T exhibited 99.97, 98.95, 98.83, 98.82, 98.75 and less than 98.7% sequence similarities to 'Streptomyces dioscori' A217, Streptomyces ederensis JCM 4958T, Streptomyces glomeroaurantiacus NBRC 15418T, Streptomyces aurantiacus NBRC 13017T, Streptomyces umbrinus JCM 4521T and other species with validly published names in the genus Streptomyces . However, the digital DNA–DNA relatedness and average nucleotide identity values between ZYC-3T, BH-SS-21T, and their closely related strains were significantly lower than the recommended threshold values. Also, phenotypic, chemotaxonomic and genetic features distinguished ZYC-3T and BH-SS-21T from their reference strains. On the basis of their genotypic and phenotypic characteristics, strains ZYC-3T and BH-SS-21T were classified as representing novel species of the genus Streptomyces under the names Streptomyces liliifuscus sp. nov. ZYC-3T (=CICC 25040T=JCM 34560T=MCCC 1K04978T) and Streptomyces liliiviolaceus sp. nov. BH-SS-21T (=MCCC 1K06236T=JCM 34767T), respectively.

The ABCC2 protein of Plutella xylostella is an important target of Cry1A toxins from Bacillus thuringiensis (Bt), but whether this protein is involved in the resistance of P. xylostella to other insecticides remains unclear. In this study, the abcc2 gene of P. xylostella was cloned and the expression levels of Pxabcc2 in susceptible and resistant strains were investigated. ABCC2 was found to be expressed 3.2-6.7-fold higher in the resistant strain than in the susceptible strain; in the surviving P. xylostella, ABCC2 levels were significantly higher when treated with indoxacarb, avermectin, and beta-cypermethrin. We constructed a stable ABCC2-expressing HEK-293 cell line to reveal the contribution of ABCC2 to insecticide resistance. The avermectin and chlorfenapyr sensitivities of the stably-transfected cell line were significantly lower than those of the control cells. The intracellular avermectin concentration was significantly lower in the stably-transfected cell line than in the control cells after four hours of exposure. This study shows that up-regulated ABCC2 expression is related to insecticide resistance in P. xylostella. Moreover, we used RNA interference technology to reduce ABCC2 levels in P. xylostella. Down-regulating ABCC2 expression did not significantly affect avermectin or chlorfenapyr resistance in P. xylostella. We speculate that increased ABCC2 expression can enhance metabolic resistance in P. xylostella. This study also provides new insights into cross-resistance between B. thuringiensis toxins and chemical insecticides.

Trehalase regulates energy metabolism in insects by converting trehalose into two glucose molecules. High amounts of trehalase are critical for insect flight and larval stress resistance. However, whether trehalase participates in the development of pesticide resistance remains unclear. In this study, we explored this phenomenon and the mechanism that underlies the regulation of

The large-conductance calcium-activated potassium channel (BKCa ) plays an important role in the regulation of insect neural circuits and locomotion, and thus is a potential target of insecticides. In this study, iberiotoxin, an inhibitor of BKCa , was found to prolong the anesthetic time of ethyl acetate on Plutella xylostella larvae. Therefore, the coding sequence of slowpoke gene coding the alpha subunit of BKCa was cloned to investigate the function of this channel in P. xylostella, and the gene expression profile in the developmental stages and tissues was also characterized. The total length of pxslo DNA was more than 19.9 kb, which harbored four alternative splicing sites (ASP-A, ASP-C, ASP-E, and ASP-G), and the coding sequence of pxslo with the highest frequency of splicing (GenBank ID: MN938456) was 3,405 base pair. The characterized PxSlo protein contained conserved domains previously identified in other insects. Quantitative reverse transcription-polymerase chain reaction analysis showed that pxslo was expressed in all the developmental stages of P. xylostella, with the highest level in adults. In the larval stage, pxslo was mainly expressed in the head and epidermis, while a limited protein was expressed in the midgut. In the adult stage, pxslo was highly expressed in the head, followed by in the ovarian tubule, and was not expressed in the testis or wings. These results suggest that BKCa plays an important physiological role in P. xylostella and provides useful information for the functional study and screening of BKCa inhibitors.

Tyramine receptors (TARs) can be activated by tyramine (TA) or octopamine (OA) and have been shown to be related to physiological regulation (e.g., gustatory responsiveness, social organization, and learning behavior) in a range of insect species. A tyramine receptor gene in Plutella xylostella, Pxtar1, was cloned and stably expressed in the HEK-293 cell line. Pharmacological properties and expression profile of Pxtar1 were also analyzed. Tyramine could activate the PxTAR1 receptor, increasing the intracellular Ca2+ concentration ((Ca2+)i) at an EC50 of 13.1 nM and reducing forskolin (10 μM)-stimulated intracellular cAMP concentration ((cAMP)i) at an IC50 of 446 nM. DPMF (a metabolite of amitraz) and L(-)-carvone (an essential oil) were found to act as PxTAR1 receptor agonists. Conversely, yohimbine and mianserin had significant antagonistic effects on PxTAR1. In both larvae and adults, Pxtar1 had the highest expression in the head capsule and expression of Pxtar1 was higher in male than in female reproductive organs. This study reveals the temporal and spatial differences and pharmacological properties of Pxtar1 in P. xylostella and provides a strategy for screening insecticidal compounds that target PxTAR1.

Apoptotic protease activating factor-1 (Apaf-1) is an adaptor molecule, essential for activating initiator caspase and downstream effector caspases, which directly cause apoptosis. In fruit flies, nematodes, and mammals, Apaf-1 has been extensively studied. However, the structure and function of Apaf-1 in Lepidoptera remain unclear. This study identified a novel Apaf-1 from Spodoptera litura, named Sl-Apaf-1. Sl-Apaf-1 contains three domains: a CARD domain, as well as NOD and WD motifs, and is very similar to mammalian Apaf-1. Interference of Sl-apaf-1 expression in SL-1 cells blocked apoptosis induced by actinomycin D. Overexpression of Sl-apaf-1 significantly enhances apoptosis induced by actinomycin D in Sf9/SL-1/U2OS cells, suggesting that the function of Sl-Apaf-1 is evolutionarily conserved. Furthermore, Sl-Apaf-1 could interact with Sl-caspase-5 (a homologue of mammalian caspase-9) and yielded a binding affinity of 1.37 &times, 106 M&ndash, 1 according isothermal titration calorimetry assay. Initiator caspase (procaspase-5) of S. litura could be activated by Sl-Apaf-1 (without WD motif) in vitro, and the activated Sl-caspase-5 could cleave Sl-procaspase-1 (a homologue of caspase-3 in mammals), which directly caused apoptosis. This study demonstrates the key role of Sl-Apaf-1 in the apoptosis pathway, suggesting that the apoptosis pathway in Lepidopteran insects and mammals is conserved.

In insects, inward-rectifying potassium (Kir) channels regulate vital physiological functions, such as feeding behavior, silk secretion, renal excretion, and immune function. Therefore, they offer promising potential as targets for insecticides. Three types of Kir subunits have been identified in Diptera and Hemiptera, but the Kir subunits of Lepidoptera still remain unclear. This study identified five Kir subunit genes (pxkir1, pxkir2, pxkir3A, pxkir3B, and pxkir4) in the transcriptome of Plutella xylostella. Phylogenetic analysis identified pxkir1, pxkir2, pxkir3A, and pxkir3B as orthologous genes of kir1&ndash, 3 in other insects. Interestingly, pxkir4 may be encoding a new class of Kir subunit in Lepidoptera that has not been reported to date. To identify further Kir channel subunits of P. xylostella, the gene expression profiles of five pxkir genes were studied by quantitative real-time PCR. These pxkir genes are expressed throughout the development of P. xylostella. pxkir1 and pxkir2 were highly expressed in thoraxes and legs, while pxkir3 (3A and 3B) and pxkir4 had high expression levels in the midgut and Malpighian tubules. This study identified the composition and distribution of Kir subunits in P. xylostella for the first time, and provides useful information for the further study of Kir channel subunits in Lepidoptera.

The β-adrenergic-like octopamine receptor (OA2B2) belongs to the class of G-protein coupled receptors. It regulates important physiological functions in insects, thus is potentially a good target for insecticides. In this study, the putative open reading frame sequence of the Pxoa2b2 gene in Plutella xylostella was cloned. Orthologous sequence alignment, phylogenetic tree analysis, and protein sequence analysis all showed that the cloned receptor belongs to the OA2B2 protein family. PxOA2B2 was transiently expressed in HEK-293 cells. It was found that PxOA2B2 could be activated by both octopamine and tyramine, resulting in increased intracellular cyclic AMP (cAMP) levels, whereas dopamine and serotonin were not effective in eliciting cAMP production. Further studies with series of PxOA2B2 agonists and antagonists showed that all four tested agonists (e.g., naphazoline, clonidine, 2-phenylethylamine, and amitraz) could activate the PxOA2B2 receptor, and two of tested antagonists (e.g., phentolamine and mianserin) had significant antagonistic effects. However, antagonist of yohimbine had no effects. Quantitative real-time polymerase chain reaction analysis showed that Pxoa2b2 gene was expressed in all developmental stages of P. xylostella and that the highest expression occurred in male adults. Further analysis with fourth-instar P. xylostella larvae showed that the Pxoa2b2 gene was mainly expressed in Malpighian tubule, epidermal, and head tissues. This study provides both a pharmacological characterization and the gene expression patterns of the OA2B2 in P. xylostella, facilitating further research for insecticides using PxOA2B2 as a target.

The cotton bollworm, Helicoverpa armigera, has developed strong resistance to many insecticides. Sterol Carrier Protein-2 (SCP-2) is an important non-specific lipid transfer protein in insects and appears to be a potential new target. In order to elucidate the structure and function of Helicoverpa armigera SCP-2 (HaSCP-2), NMR spectroscopy, docking simulations, mutagenesis and bioassays were performed. HaSCP-2 composed of five α-helices and four stranded β-sheets. The folds of α-helices and β-sheets interacted together to form a hydrophobic cavity with putative entrance and exit openings, which served as a tunnel for accommodating and transporting of lipids. Several sterols and fatty acids could interact with HaSCP-2 via important hydrophobic sites, which could be potential targets for insecticides. Mutagenesis experiments indicated Y51, F53, F89, F110, I117 and Q131 may be the key functional sites. HaSCP-2 showed high cholesterol binding activity and SCP-2 inhibitors (SCPIs) could inhibit the biological activity of HaSCP-2. SCPI-treated larvae at young stage showed a significant decrease of cholesterol uptake in vivo. Our study describes for the first time a NMR structure of SCP-2 in lepidopteran H. armigera and reveals its important function in cholesterol uptake, which facilitates the screening of effective insecticides targeting the insect cholesterol metabolism.

Cholesterol is a membrane component and the precursor of ecdysteroids in insects, but insects cannot synthesize cholesterol de novo. Therefore, cholesterol uptake and transportation during the feeding larval stages are critical processes in insects. The sterol carrier protein-2 domain (SCP-2) in sterol carrier proteins-x (SCP-x) has been speculated to be involved in intracellular cholesterol transfer and metabolism in vertebrates. However, a direct association between SCP-x gene expression, cholesterol absorption and development in lepidopteran insects is poorly understood. We identified the Helicoverpa armigera sterol carrier protein-x/2 (HaSCP-x/2) gene from the larval midgut cDNAs. The HaSCP-x/2 gene is well conserved during evolution and relatively divergent in heterogenetic species. Transcripts of HaSCP-x/2 were detected by qRT-PCR at the highest level in the midgut of H. armigera during the larval stages. Expression knockdown of HaSCP-x/2 transcripts via dsRNA interference resulted in delayed larval development and decreased adult fecundity. Sterol carrier protein-2 inhibitors were lethal to young larvae and decreased fertility in adults emerged from treated elder larvae in H. armigera. The results taken together suggest that HaSCPx/2 gene is important for normal development and fertility in H. armigera.