RNA Interference of Phenoloxidases of the Fall Armyworm, Spodoptera frugiperda, Enhance Susceptibility to Bacillus thuringiensis Protein Vip3Aa19.

ABSTRACT

Phenoloxidase (PO) is an important enzyme in the cellular immune system and is involved in defense against a wide range of pathogens, including Bacillus thuringiensis. Vip3Aa19 is secreted and expressed by Bacillus thuringiensis (Bt) at the middle exponential growth phase and is a kind of protein with efficient insecticidal activity against Spodoptera frugiperda. However, immune responses of the target insects have been regarded as a hindrance to Bt pathogenicity. This paper reports two phenoloxidase (PO) genes (SfPAE and SfPO2) identified from the hemocyte transcriptome data of the fall armyworm, Spodoptera frugiperda. qRT-PCR validation results showed that the expression levels of two PO genes were significantly upregulated after Vip3Aa19 (LC50 = 4.98 µg/g) toxin treatment compared with those of S. frugiperda fed an insecticide-free artificial diet. Meanwhile, two PO genes were expressed from the egg to adult stages even without an immune challenge. We noticed that at all developmental stages investigated in the S. frugiperda, SfPAE was generally expressed at a higher level than SfPO2. However, after Vip3Aa19 treatment, the SfPO2 gene mRNA expression level was significantly elevated in response to the toxin challenge. An injection of a specific double-stranded RNA (dsRNA) against POs could suppress its expression. The third instar larvae of S. frugiperda treated with dsRNA were much more susceptible to Vip3Aa19 toxin than the control larvae were. Notably, the mortality rate was nearly 90% after a dsPO2 injection. These results proved that SfPO2 was more important for the survival of S. frugiperda. Finally, RNA interference and then PO activity detection revealed that PO genes mainly existed in the hemolymph and played an important role in immune defense against Bt toxin.