Deletion mutant of sPLA2 using CRISPR/Cas9 exhibits immunosuppression, developmental retardation, and failure of oocyte development in legume pod borer, Maruca vitrata

Abstract

Phospholipase A2 (PLA2) catalyzes release of free fatty acids linked to phospholipids at sn-2 position. Some of these released free fatty acids are used to synthesize eicosanoids that mediate various physiological processes in insects. Although a large number of PLA2s form a superfamily consisting of at least 16 groups, few PLA2s have been identified and characterized in insects. Furthermore, physiological functions of insect PLA2s remain unclear. Clustered regularly interspaced short parlindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) has been a useful research tool to validate gene function. This study identified and characterized a secretory PLA2 (sPLA2) from legume pod borer, Maruca vitrata (Lepidoptera: Crambidae), and validated its physiological functions using CRISPR/Cas9. An open reading frame of M. vitrata sPLA2 (Mv-sPLA2) encoding 192 amino acids contained signal peptide, calcium-binding domain, and catalytic site. Phylogenetic analysis indicated that Mv-sPLA2 was related to other Group III sPLA2s. Mv-sPLA2 was expressed in both larval and adult stages. It was inducible by immune challenge. RNA interference (RNAi) of Mv-sPLA2 significantly suppressed cellular immunity and impaired larval development. Furthermore, RNAi treatment in female adults prevented oocyte development. These physiological alterations were also observed in a mutant line of M. vitrata with Mv-sPLA2 deleted by using CRISPR/Cas9. Mv-sPLA2 was not detected in the mutant line from western blot analysis. Addition of an eicosanoid, PGE2, significantly rescued oocyte development of females of the mutant line. These results suggest that Mv-sPLA2 plays crucial role in immune, developmental, and reproductive processes of M. vitrata.