Research Article Received: 6 December 2019 Revised: 1 August 2020 Accepted article published: 31 August 2020 Published online in Wiley Online Library: (wileyonlinelibrary.com) DOI 10.1002/ps.6066 Functional analysis of an upregulated calmodulin gene related to the acaricidal activity of curcumin against Tetranychus cinnabarinus (Boisduval) Hong Zhou,† Fuyou Guo,† Jinxiang Luo, Yongqiang Zhang, Jinlin Liu, Yanchun Zhang, Xinyu Zheng, Fenglin Wan and Wei Ding* Abstract BACKGROUND: Curcumin is a promising botanical acaricidal compound with activity against Tetranychus cinnabarinus. Calmod- ulin (CaM) is a key calcium ion (Ca2+) sensor that plays a vital role in calcium signaling. Overexpression of the CaM gene with inducible character occurs in curcumin-treated mites, but its functional role remains to be further analyzed by RNA interference (RNAi) and protein expression. RESULTS: A CaM gene was cloned from T. cinnabarinus (designated TcCaM). TcCaM was upregulated and the protein was acti- vated in mites by curcumin. The susceptibility of mites to curcumin was decreased after inhibiting CaM function with anti- CaM drug trifluoperazine (TFP) and silencing CaM transcription with RNAi, suggesting that the CaM gene is involved in the acar- icidal activity of curcumin against mites. Moreover, the TFP pre-treated Sf9 cells were resistant to curcumin-mediated increase in [Ca2+]i levels, indicating that CaM-mediated Ca2+ homeostasis was disturbed by curcumin. TcCaM was then re-engineered for heterologous expression in Escherichia coli. Strikingly, our results showed that the recombinant CaM protein was directly acti- -1 vated by curcumin via inducing its conformational changes, its half-maximal effective concentration (EC50) value is 0.3 ∼mol L in vitro, which is similar to curcumin against CaM-expressing Sf9 cells (0.76 ∼mol L-1) in vivo. CONCLUSION: These results confirm that the overexpressed CaM gene is involved in the acaricidal activity of curcumin, and the mode of action of curcumin may be via activating CaM function, and thereby disrupting Ca2+ homeostasis in T. cinnabarinus. This study highlights the novel target mechanism of new acaricides, promoting our understanding of the molecular mechanism of CaM-mediated acaricide targets in mites. © 2020 Society of Chemical Industry Supporting information may be found in the online version of this article. Keywords: Tetranychus cinnabarinus; curcumin; calmodulin; RNAi; heterologous expression addition, studies have confirmed that curcumin has broad- 1 INTRODUCTION 7 The carmine spider mite, Tetranychus cinnabarinus, is one of the prin- spectrum pharmacological activities, such as acaricidal, insecticidal,8 antifungal,9 anti-inflammatory,10 antioxidant,11 cipal global agricultural mite pests of arthropods, feedings on over 12 13 100 types of plants, such as cotton, beans, corn, wheat, sesame, sun- anti-Alzheimer's, and anticarcinogenic effects. In particular, 1–3 our previous research discovered that curcumin has outstanding flower, and tomato. These mites are extremely difficult to prevent repellent, contact killing, and ovipositional inhibition effects and control because of their high fertility and inbreeding rate, small against T. cinnabarinus.7, 14 Moreover, several characteristic neuro- size, short developmental duration, and strong pesticide resistance.4 toxicity symptoms, including excitement and spasm, were Thus far, control of T. cinnabarinus mainly relies on chemical acari- observed in mites exposed to curcumin, suggesting that cides or insecticides, including pyridaben, spiromesifen, etoxazole and avermectin. However, there are many issues associated with acaricides, including the development of resistance, threats to non- * Correspondence to: W Ding, College of Plant Protection, Southwest University, target organisms, and environmental contamination. Consequently, Chongqing 400715, P. R. China. E-mail: [email protected] an environmentally friendly acaricidal compound with a novel target † should be identified and advanced to address these issues. These authors contributed equally. Curcumin is extracted from the rootstock of the plant Curcuma longa and extensively used as a food preservative, colorant, spice, Institute of Pesticide Science, College of Plant Protection, Southwest University, and additive in cosmetics and pharmaceutical preparations.5, 6 In Chongqing, P. R. China 1 Pest Manag Sci 2020 www.soci.org © 2020 Society of Chemical Industry www.soci.org H Zhou et al. curcumin is a neurotoxin for which internal cellular calcium ion 2.2 [Ca2+]i assay (Ca2+) acts a vital role as a second messenger.15 We also investi- Sf9 (Spodoptera frugiperda) cells were maintained at 27 ± 1 °Cin gated the transcriptome of T. cinnabarinus after exposure to cur- Sf-900™ SFM culture medium (Gibco, California, USA). Because cumin. Interestingly, a gene expressing calmodulin (CaM), which the cell lines of mites cannot be cultured in vitro, Sf9 cells were may participate in the action model of curcumin, was significantly used for experiments and treated with curcumin (99%, Shanghai upregulated in T. cinnabarinus.16 However, the functional roles of Yuanye Biotechnology, Shanghai, China) at concentrations of μ -1 CaM in the acaricidal mechanism of curcumin remain unclear in 0, 2.6, 8.2 and 53 mol L (LC30,LC50 and LC80) for 48 h to detect 2+ T. cinnabarinus. the effect of curcumin on internal cellular [Ca ]i levels. Fluo-4/AM Calcium ions are central signaling molecules in neurons that fluorescence staining (Beyotime Biotechnology, Shanghai, China) 2+ regulate various biological functions, including neurotransmitter was used to measure [Ca ]i concentrations in the Sf9 cells, and 2+ 26 release, gene expression, and muscle contraction.17 Many cyto- the details of the [Ca ]i assay were described by Zhou et al. plasmic Ca2+-binding proteins mediate the intracellular actions of Ca2+.18 Among these Ca2+-binding proteins, CaM is the most 2.3 Bioassay important; CaM is principally expressed in the brain and acts a Curcumin toxicity against T. cinnabarinus was determined by the 27, 28 vital role in various physiological functions such as gene transcrip- FAO-recommended slip-dip method. The sublethal concen- tion, muscle contraction and relaxation, and signal transduction.19 trations of curcumin against mites were measured by log-probit 29 Although CaM itself does not display any catalytic activity, it reg- analysis of bioassay data. Each independent experiment con- ulates the activity of numerous CaM-dependent enzymes, includ- sisted of three biological replicates. In the bioassay, the LC10, 2+ 2+ ing phosphodiesterases (PDE), Ca -Mg -ATPase, protein LC30, and LC50 values of curcumin were 0.21, 1.23, and 2.64 mg -1 kinases, phosphatases, and nitric oxide synthases (NOSs).20 CaM L , respectively. consists of approximately symmetrical N- and C-terminal lobes TcCaM (hereafter referred to as the N- and C-lobes) linked by a flexible 2.4 Total RNA extraction, cDNA synthesis, and 21, 22 2+ cloning helix. Each lobe can jointly bind two Ca in the micromolar – range via two EF-hand motifs.23 Upon Ca2+ binding to either lobe, Female adult (3 5 days old) mites were used to extract total RNA several hydrophobic residues exposed promote the interaction of with the RNeasy® plus Micro Kit (Tiangen, Beijing, China). The RNA CaM with a target sequence of a regulated enzyme.23 Notably, evi- quantity was determined at OD260/280 using a Nanovue UV-visible fi dence has shown that CaM can be considered a potential target spectrophotometer (GE Healthcare, Fair eld, CT, USA) and further for pest management; e.g. the insecticidal mechanism of toma- validated by 1% agarose gel electrophoresis. Complementary tine against Helicoverpa armigera is mediated by CaM,24 and spe- DNA (cDNA) was synthesized using PrimeScript® 1st Strand cDNA cific antagonists of CaM mediated by trifluoperazine (TFP, a Synthesis Kit (Takara, Dalian, China). The cDNA was stored at − ° specific inhibitor of CaM) inhibit the production of sex phero- 20 C. fi mones in Bombyx mori.25 Speci c primers (Supporting Information Table S1) were Better characterization of the molecular targets of curcumin designed and synthesized to obtain the DNA sequence of the in mites may help in gaining insight about the mode of action CaM gene on the basis of the whole-genome sequence of the sis- of curcumin and aid in the discovery and development of ter species T. urticae. Polymerase chain reactions (PCRs) were con- × μ μ μ new acaricides with novel targets, such as CaM in mites, for ducted with 10 PCR buffer (2.5 L), MgCl2 (2.5 L), dNTPs (2.0 L), μ ™ μ pest management. Here, the identification and verification of cDNA template (1 L), rTaq polymerase (0.2 L), F/R primers μ μ fi CaM acting as a molecular target of curcumin in mites are (1 L), and 14.8 L doubled-distilled water (ddH2O) to a nal reac- μ ° described. We first sequenced and then phylogenetically ana- tion volume of 25 L. The PCR program consisted of 94 C for ° ° ° lyzed and characterized the expression pattern of a gene 3 min, 35 cycles of 94 C for 30 s, 55 C for 30 s, and 72 C for fi ° expressing CaM; the interaction between curcumin and CaM 50 s, and a nal extension for 10 min at 72 C. The PCR products fi was subsequently investigated. A reverse genetic study using were gel-puri ed using a Gel Extraction Mini Kit (Tiangen), ligated ™ leaf-mediated double-stranded RNA (dsRNA) feeding was then into the pMD 19-T vector (Takara), and transfected into Escheri- ⊍ employed to examine the link between the CaM gene and the chia coli DH5 -competent cells (Tiangen) for sequencing at the acaricidal mechanism of curcumin against T. cinnabarinus;a Beijing Genomics Institute (Beijing, China). functional cytotoxicity assay using Sf9 cells overexpressing CaM was also performed. The current research first shows that 2.5 Gene characteristic and phylogenetic analysis The CaM gene sequence was edited with DNAMAN 5.2.2, and curcumin is a novel agonist of CaM function and offers pivotal 30,31 clues for elucidating the molecular mechanisms underlying amino acid sequences were aligned via the ClustalW program.