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.
Highlights
IntroductionInsects have a potent and efficient innate immune system
Like many invertebrates, insects have a potent and efficient innate immune system
The complete sequence of serpin-4 cDNA contained a 59-untranslated region (UTR) of 312 bp, a 39-UTR of 895 bp, and an open reading frame (ORF) of 1239 bp encoding a polypeptide of 412 amino acid residues (Fig. 1)
Summary
Insects have a potent and efficient innate immune system This is considered to constitute an evolutionarily defense strategy, including cellular and humoral immunity that protects the host from infection by other organisms in a non-specific manner [1]. The innate immune processes including body melanization, blood coagulation, cell encapsulation, phagocytosis, complement activation, and synthesis of antimicrobial peptides are regulated by the protease cascades resulting in multiple steps of protease activation [2,3,4]. Several intracellular as well as extracellular serpins have been identified in the insect genome [12,13,14], with most of these being reported to participate in the regulation of innate immune responses such as modulating prophenoloxidase (pro-PO) activation, hemolymph coagulation and synthesis of antimicrobial peptides (AMPs) [15]. All of them could inhibit and regulate proteases that lead to activation of the pro-PO system and the cytokine spatzle function in cascades [19,22,23,24,25,26,27,28,29,30]
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