Biosynthesis and immunity of epoxyeicosatrienoic acids in a lepidopteran insect, Spodoptera exigua.

Eicosanoids mediate both cellular and humoral immune responses in insects. Epoxyeicosatrienoic acids (EETs) are a group of eicosanoids containing epoxide formed by epoxygenase (EPX) activity of cytochrome P450 (CYP). Although EETs have been considered to mediate immune responses in some insects, their synthetic machinery was little understood in insects. This study monitored EETs in a lepidopteran insect, Spodoptera exigua, immunized with bacteria and found all four EETs (5,6-EET, 8,9-EET, 11,12-EET, and 14,15-EET) from larval fat body at 247-1,736 pg/g levels. Then to predict EPXs, 140 CYPs were collected from S. exigua transcriptomes and compared with human EPXs. Four CYPs (SeEPX1-SeEPX4) sharing homologies with human EPXs were chosen and assessed in subsequent expression and functional analyses. All four EPXs were expressed in all development stages. In larval stage, all four EPXs were expressed in immune-associated tissues such as fat body and hemocytes. Furthermore, their expression levels were highly enhanced by bacterial challenge in different tissues. RNA interference (RNAi) using gene-specific double stranded RNA injection suppressed their expression levels by more than 55%. RNAi treatments interfered with hemocyte-spreading behavior and nodule formation upon bacterial challenge except RNAi treatment against SeEPX2. All four EETs stimulated cellular immune response measured by nodule formation in S. exigua. The suppressed immune responses by the RNAi treatments against three SeEPXs were rescued by the addition of 8,9-EET. However, other three EETs gave their specific rescue effect depending on SeEPX types under RNAi. In humoral immune response, all four RNAi treatments suppressed expression of antimicrobial peptide genes. This study reports the presence of all four EETs in larval fat body of S. exigua and suggests that four SeEPXs are associated with immune responses mediated by EETs.