A novel calcium-independent cellular PLA2 acts in insect immunity and larval growth

Phospholipase A(2) (PLA(2)) catalyzes the position-specific hydrolysis of fatty acids linked to the sn-2 position of phospholipids (PLs). PLA(2)s make up a very large superfamily, with more than known 15 groups, classified into secretory PLA(2) (sPLA(2)), Ca2+-dependent cellular PLA(2) (sPLA(2)) and Ca2+-independent cellular PLA(2) (iPLA(2)). Only a few insect sPLA(2)s, expressed in venom glands and immune tissues, have been characterized at the molecular level. This study aimed to test our hypothesis that insects express iPLA(2), using the beet armyworm, Spodoptera exigua, our model insect. Substantial PLA(2) activities under calcium-free condition were recorded in several larval tissue preparations. The PLA(2) activity was significantly reduced in reactions conducted in the presence of a specific IPLA(2) inhibitor, bromoenol lactone (BEL). Analysis of a S. exigua hemocyte transcriptome identified a candidate iPLA(2) gene (SeiPLA(2)-A). The open reading frame encoded 816 amino acid residues with a predicted molecular weight of 90.5 kDa and 6.15 pI value. Our phylogenetic analysis clustered SeiPLA(2)-A with the other vertebrate iPLA(2)s. SeiPLA(2)-A was expressed in all tissues we examined, including hemocytes, fat body, midgut, salivary glands, Malpighian tubules and epidermis. Heterologous expression in Sf9 cells indicated that SeiPLA(2)-A was localized in cytoplasm and exhibited significant PLA(2) activity, which was independent of Ca2+ and inhibited by BEL. RNA interference (RNA of SeiPLA(2)-A using its specific dsRNA in the fifth instar larvae significantly suppressed iPLA(2) expression and enzyme activity. dsSeiPLA(2)-A-treated larvae exhibited significant loss of cellular immune response, measured as nodule formation in response to bacterial challenge, and extended larval-to-pupal developmental time. These results support our hypothesis, showing that SeiPLA(2)-A predicted from the transcriptome analysis catalyzes hydrolysis of fatty acids from cellular PLs and plays crucial physiological roles in insect immunity and larval growth. (C) 2015 Elsevier Ltd. All rights reserved.