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The carmine spider mite, Tetranychus cinnabarinus (Boisduval), is a polyphagous agricultural pest with an extensive host plant range. Scopoletin is a promising acaricidal compound whose acaricidal mechanism may occur by disrupting intracellular Ca 2+ homeostasis and calcium signaling pathways. However, the underlying mechanism of scopoletin for specific target locations of T. cinnabarinus remains unclear. In this study, a full-length cDNA of the L-type voltage-gated calcium channel (TcLTCC) subunit gene from T. cinnabarinus was cloned and characterized. The expression pattern of the TcLTCC gene in all developmental stages of T. cinnabarinus was analyzed. The gene was highly expressed in larval and nymphal stages and was significantly upregulated after treatment with scopoletin. Knocking down the TcLTCC transcript reduced the sensitivity of T. cinnabarinus to scopoletin. Homology modeling and molecular docking were also conducted. The interaction between scopoletin and TcLTCC showed that scopoletin inserted into the cavity bound to the site of the TcLTCC protein by the driving force of hydrogen bonding. This study provides insights into the mechanism by which scopoletin interacts with TcLTCC. Results can improve the understanding of the toxicity of scopoletin to T. cinnabarinus and provide valuable information for the design of new LTCC inhibitors., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The carmine spider mite, Tetranychus cinnabarinus (Boisduval), is an economically important agricultural pest that is difficult to prevent and control. Scopoletin is a botanical coumarin derivative that targets Ca 2+ -ATPase to exert a strong acaricidal effect on carmine spider mites. In this study, the full-length cDNA sequence of a plasma membrane Ca 2+ -ATPase 1 gene ( TcPMCA1 ) was cloned. The sequence contains an open reading frame of 3750 bp and encodes a putative protein of 1249 amino acids. The effects of scopoletin on TcPMCA1 expression were investigated. TcPMCA1 was significantly upregulated after it was exposed to 10%, 30%, and 50% of the lethal concentration of scopoletin. Homology modeling, molecular docking, and three-dimensional quantitative structure-activity relationships were then studied to explore the relationship between scopoletin structure and TcPMCA1 -inhibiting activity of scopoletin and other 30 coumarin derivatives. Results showed that scopoletin inserts into the binding cavity and interacts with amino acid residues at the binding site of the TcPMCA1 protein through the driving forces of hydrogen bonds. Furthermore, CoMFA (comparative molecular field analysis)- and CoMSIA (comparative molecular similarity index analysis)-derived models showed that the steric and H-bond fields of these compounds exert important influences on the activities of the coumarin compounds.Notably, the C3, C6, and C7 positions in the skeletal structure of the coumarins are the most suitable active sites. This work provides insights into the mechanism underlying the interaction of scopoletin with TcPMCA1 . The present results can improve the understanding on plasma membrane Ca 2+ -ATPase-mediated (PMCA-mediated) detoxification of scopoletin and coumarin derivatives in T. cinnabarinus , as well as provide valuable information for the design of novel PMCA-inhibiting acaricides., Competing Interests: The authors declare no conflict of interest.

The carmine spider mite Tetranychus cinnabarinus is a major pest of crop and vegetable plants worldwide. Previous studies have shown that scopoletin is a promising acaricidal compound against Tetranychus cinnabarinus. However, the acaricidal mechanism of scopoletin remains unclear. In the present study, 12 full-length cDNAs of chitinase (CHIT) genes from Tetranychus cinnabarinus (designated TcCHITs ) were cloned and characterized. Although TcCHITs were expressed throughout all life stages, their expression levels were significantly upregulated during the larval and nymphal stages. TcCHITs were downregulated 24 h after treatment with scopoletin and upregulated 24 h after treatment with diflubenzuron (DFB, a chitin synthesis inhibitor). Feeding double-stranded RNA effectively silenced TcCHIT transcription in Tetranychus cinnabarinus , thus increasing its susceptibility to scopoletin but reducing that to DFB. Meanwhile, TcCHIT silencing in larvae and adult resulted in an extremely low molting rate (7.3%) and high mortality rate (53.3%), respectively, compared with those in the control group. CHIT genes are closely related to arthropod survival, molting, and development in Tetranychus cinnabarinus , suggesting that acaricidal mechanisms of scopoletin and DFB may occur by inhibition and activation of CHIT gene expression, respectively. TcCHIT constitutes a possible target of scopoletin and DFB in Tetranychus cinnabarinus .

By using slide-dip bioassay method under laboratory condition, an investigation was made on the lethal effects of scopoletin, bisdemethoxycurcumin (BDMC), and their combination at an optimal mass ratio (7:6) of scopoletin to BDMC against the adult females of Tetranychus cinnabarinus Boisd. A time-dose mortality model (TDM) was established, which passed the Hosmer-Lemeshow test. The sensitivity of the adult females to the concentration change of the acaricides was in the sequence of scopoletin > optimal mass ratio of scopoletin to BDMC > BDMC. The peak mortality of the female adults was found at 32, 28 and 32 h after treated with BDMC, scopoletin, and their combination at the optimal mass ratio, respectively. The values of the LC50 and LC90 at 48 h after treated with BDMC, scopoletin, and their combination at the optimal mass ratio were 0.3324, 0.2035 and 0.2195 mg x mL(-1), and 2.1198, 0.9521 and 1.1617 mg x mL(-1), and the median lethal time (LT50) of BDMC, scopoletin, and their combination at the optimal mass ratio was 7.4, 6.0 and 6.1 h at the concentration 1.0 mg x mL(-1), and 6.4, 4.8 and 5.0 h at the concentration 2.0 mg x mL(-1), respectively. The acaricidal activity and time-dose response of the optimal combination of scopoletin and BDMC were closer to those of scopoletin, suggesting a synergistic acaricidal activity of the combination of scopoletin and BDMC, which was worthy to be developed for application.

This paper determined the contact-killing and fumigant activities of natural plant product bisdemethoxycurcumin (BDMC) on the important phytophagous mite Tetranychus cinnabarinus (Acari:Tetranychidae) at its different life stages, and studied the repellency effects of BDMC on the mite larvae, nymphs, and adults, and the inhibition efficiency of BDMC on the female mite oviposition under the conditions of 26 degrees C +/- 1 degree C, 60%-80% RH, and light cycle 14L : 10D h. The median lethal concentration (LC50) of BDMC at 48 h against female adults determined by slide-dip method was 0.433 mg x mL(-1). At concentration 0.883 mg x mL(-1) (LC70), the contact-killing activity of BDMC against different life stage T. cinnabarinus was in the order of larva > nymph > adult > egg, and the corrected mortality of larvae at 24 h and 48 h was 60.0% and 83.3%, respectively. BDMC had no obvious fumigant activity against different life stage T. cinnabarinus, and the corrected mortality was all less than 3% after treatment 24 h and 48 h. BDMC had stronger repellency activity against the mite, with the repellency rate against larvae at different treatment times all above 85%, followed by against nymphs, and that against adults after 72 h being only 47.8%. BDMC had obvious oviposition inhibition activity against female adults, with the inhibition rate after 120 h reached 89.3%. All the results suggested that the main action modes of BDMC against T. cinnabarinus were contact-killing, repellency, and oviposition inhibition.

Scopoletin is a promising acaricidal botanical natural compound against Tetranychus cinnabarinus , and its acaricidal mechanism maybe involve calcium overload according to our previous study. To seek potential candidate target genes of calcium overload induced by scopoletin in T. cinnabarinus , RNA-seq was utilized to detect changes in transcription levels. 24 and 48 h after treatment, 70 and 102 differentially expressed genes were obtained, respectively. Target genes included 3 signal transduction genes, 4 cell apoptosis genes, 4 energy metabolism genes, and 2 transcription factor genes. The role of 3 calcium signaling pathway-related genes, namely, G-protein-coupled neuropeptide receptor, Bcl-2 protein and guanylate kinase (designated TcGPCR , TcBAG , and TcGUK , respectively) in the calcium overload were investigated in this study. RT-qPCR detection showed that scopoletin treatment upregulated the expression level of TcGPCR and downregulated the expression level of TcBAG and TcGUK. The result of RNAi indicated that downregulation of TcGPCR decreased susceptibility to scopoletin, and downregulation of TcBAG and TcGUK enhanced susceptibility to scopoletin. Functional expression in Chinese hamster ovary cells showed that scopoletin induced a significant increase in intracellular free calcium [Ca2+]i levels by activating TcGPCR. These results demonstrated that the acaricidal mechanism of scopoletin was via disrupting intracellular Ca2+ homeostasis and calcium signaling pathway mediated by GPCR, BAG, and GUK. [ABSTRACT FROM AUTHOR]

Abstract: Various solvent extracts from different organs of Phytolacca americana L. were tested for their acaricidal activities against female carmine spider mite [Tetranychus cinnabarinus (Boisduval)] adults by slide-dip bioassay. The acetone extracts of roots with mortality rates of 95.72% and 100.00% at 2.5mg/mL were more active than those of leaves and stems and compared with other solvent extracts from other organs after 48 and 72h of treatment, respectively. The root acetone extracts were fractionated using a silica gel column; after thin-layer chromatography, the concentrated extracts were separated into 15 groups and further analyzed for their acaricidal activities. The ninth fraction exhibited the highest acaricidal activity. Phytochemical evaluation and liquid chromatography/mass spectrometry analysis revealed that esculentoside P was the dominant active phytolaccasaponin in the said fraction. This biological compound is a promising candidate as an acaricide against T. cinnabarinus. [Copyright &y& Elsevier]