RNAi-Mediated Knockdown of Serine Protease Inhibitor Genes Increases the Mortality of Plutella xylostella Challenged by Destruxin A

Abstract

Destruxin A is a mycotoxin that is secreted by entomopathogenic fungi which has a broad-spectrum insecticidal effect. Previous transcript and protein profiling analysis showed that destruxin A has significant effects on the expression of serine protease inhibitor genes (serpin-2, 4, 5) in the larvae of Plutella xylostella. In the current study, we aimed to understand the role of serpins under application of destruxin A. We obtained two full-length cDNA sequences of P. xylostella serpins, named serpin-4 and serpin-5, and cloned the serpin-2 gene whose full-length has already been published. Phylogenetic analysis indicated that these two serpin genes were highly clustered with other serpins associated with the immune response in other insects. The temporal and spatial expression of serpin-2, serpin-4 and serpin-5 were determined to be the highest in the fat body and hemolymph of 4th larval stage using qRT-PCR and western blot detection techniques. RNA interference (RNAi) mediated knockdown of P. xylostella serpin genes was carried out by microinjection of double-stranded RNA (dsRNA). The expression levels of serpins decreased significantly after RNAi. Results showed that the depletion of serpins induced cecropins expression, increased phenoloxidase (PO) activity, body melanization and mortality in the larvae of P. xylostella under the same lethal concentration of destruxin A. The superimposed effects of serpins RNAi were similar with the destruxin A treatment upon mortality of P. xylostella larvae. We discovered for the first time that serpins play indispensable role in P. xylostella when challenged by destruxin A and deduced the possible function mechanism of destruxin A. Our findings are conducive to fully understanding the potential insecticidal mechanism of destruxin A and constitute a well-defined potential molecular target for novel insecticides.

Collaborative contribution of six cytochrome P450 monooxygenase genes to fenpropathrin resistance in Tetranychus cinnabarinus (Boisduval)

“…A similar phenomenon was also observed by Han et al . (), in which at least 55.56% higher mortality was detected when three types of serpin dsRNAs were simultaneously injected into Tenebrio molitor compared with injections of either one or two of these dsRNA. What is more, insecticide resistance can also occur as a result of detoxification activity of a single P450, such as CYP6D1 in the house fly (Lin & Scott, ), or of multiple P450s, such as CYP9A12 , CYP9A14 and CYP6B7 in the cotton bollworm (Yang et al ., ).…”

Stability of cyflumetofen resistance in Tetranychus cinnabarinus and its correlation with glutathione‐S‐transferase gene expression

“…Moreover, it is worth mentioning that we could not ignore the contributions of other upregulated GST genes since six overexpressed GST genes were detected in CyR strain. The collaborative contribution against resistance from multiple up‐regulated genes had been demonstrated in resistant insects and mites . Nevertheless, the superiority of TcGSTm02 in cyflumetofen‐resistant T. cinnabarinus is interesting and important.…”

“…The collaborative contribution against resistance from multiple up-regulated genes had been demonstrated in resistant insects and mites. [37][38][39] Nevertheless, the superiority of TcGSTm02 in cyflumetofen-resistant T. cinnabarinus is interesting and important.…”

20‐Hydroxyecdysone enhances Immulectin‐1 mediated immune response against entomogenous fungus in Locusta migratoria

“…The methods of RNA extraction and cDNA cloning were as previously described. 43 A pair of specific primers LmIML-1-F and LmIML-1-R was designed based on the Unigene fragment obtained from transcriptome of the L. migratoria (Table 1), and the target gene was amplified by using the whole-body cDNA of the fifth instar nymphs. The signal peptide, protein domains and the conserved motifs were predicted by SMART tools (http://smart .embl-heidelberg.de/).…”