title: Eicosanoids up-regulate production of reactive oxygen species by NADPH-dependent oxidase in Spodoptera exigua phagocytic hemocytes.
abstract: Eicosanoids mediate cellular immune responses in insects, including phagocytosis of invading microbes. Phagocytosis entails two major steps, the internalization of microbes and the subsequent killing of them via formation of reactive oxygen species (ROS). Here, we posed the hypothesis that eicosanoids mediate ROS production by activating NADPH-dependent oxidase (NOX) and tested the idea in the model insect, Spodoptera exigua. A NOX gene (we named SeNOX4) was identified and cloned, yielding a full open reading frame encoding 547 amino acid residues with a predicted molecular weight of 63,410Da and an isoelectric point at 9.28. A transmembrane domain and a large intracellular domain containing NADPH and FAD-binding sites were predicted. Phylogenetic analysis indicated SeNOX4 clusters with other NOX4 genes. SeNOX4 was expressed in all life stages except eggs, and exclusively in hemocytes. Bacterial challenge and, separately, arachidonic acid (AA, a precursor of eicosanoid biosynthesis) injection increased its expression. The internalization step was assessed by counting hemocytes engulfing fluorescence-labeled bacteria. The phagocytic behavior was inhibited by dsRNA suppression of SeNOX4 expression and, separately by dexamethasone (DEX, a specific inhibitor of eicosanoid biosynthesis) treatments. However, injecting AA to dsSeNOX4-treated larvae did not rescue the phagocytic activity. Hemocytic ROS production increased following bacterial challenge, which was sharply reduced in dsSeNOX4-treated, and separately, in DEX-treated larvae. AA partially reversed the suppressed ROS production in dsSeNOX4-treated larvae. Treating larvae with either the ROS-suppressing dsSeNOX4 construct or DEX rendered experimental larvae unable to inhibit bacterial proliferation in their hemocoels. We infer that eicosanoids mediate ROS production during phagocytosis by inducing expression of SeNOX4.
authors.affiliation: Department of Bioresource Sciences, Andong National University, Andong 760-749, Republic of Korea.
authors.forename: Youngjin
authors.initials: Y
authors.lastname: Park
authors.affiliation: Biological Control of Insects Research Laboratory, USDA/Agricultural Research Service, 1503 Providence Rd., Columbia, MO 65203, USA.
authors.forename: David W
authors.initials: DW
authors.lastname: Stanley
authors.affiliation: Department of Bioresource Sciences, Andong National University, Andong 760-749, Republic of Korea. Electronic address: hosanna@andong.ac.kr.
authors.forename: Yonggyun
authors.initials: Y
authors.lastname: Kim
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016718:Arachidonic Acid; D003907:Dexamethasone / Q000494:pharmacology; D015777:Eicosanoids / Q000378:metabolism*; D004926:Escherichia coli; D006434:Hemocytes / Q000378:metabolism; D007111:Immunity, Cellular; D007814:Larva / Q000235:genetics / Q000276:immunology; D008969:Molecular Sequence Data; D019255:NADPH Oxidases / Q000378:metabolism; D010587:Phagocytosis; D010802:Phylogeny; D034622:RNA Interference; D017382:Reactive Oxygen Species / Q000378:metabolism*; D018411:Spodoptera / Q000235:genetics* / Q000276:immunology; D015854:Up-Regulation
chemical_list: D015777:Eicosanoids; D017382:Reactive Oxygen Species; D016718:Arachidonic Acid; D003907:Dexamethasone; D019255:NADPH Oxidases
keywords: Eicosanoids; NOX; Phagocytosis; RNA interference; ROS; Spodoptera exigua