Functional analysis of an upregulated calmodulin gene related to the acaricidal activity of curcumin against Tetranychus cinnabarinus (Boisduval).

Abstract

Curcumin is a promising botanical acaricidal compound with activity against Tetranychus cinnabarinus. Calmodulin (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. A CaM gene was cloned from T. cinnabarinus (designated TcCaM). TcCaM was upregulated and the protein was activated 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 acaricidal 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 activated by curcumin via inducing its conformational changes, its half-maximal effective concentration (EC50 ) value is 0.3 μmol L-1 in vitro, which is similar to curcumin against CaM-expressing Sf9 cells (0.76 μmol L-1 ) in vivo. 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.