Sony Shrestha

Various eicosanoids modulate the cellular and humoral immune responses of the beet armyworm, Spodoptera exigua.

Cyclooxygenase (COX) and lipoxygenase (LOX) can catalyze the oxidation of C20 fatty acids to produce certain eicosanoids, which play roles in mediating immune responses in insects. Despite their critical role in insect immunity, there have been few studies of the unique effects of different eicosanoids on immune responses. This study analyzed cellular and humoral immune responses of the beet armyworm, Spodoptera exigua, using seven eicosanoids selected from two major eicosanoid subgroups: prostaglandin (PG) and leukotriene (LT), derived from catalytic activities of COX and LOX respectively. Upon bacterial challenge, all seven eicosanoids (PGA1, PGB2, PGD2, PGE1, PGE2, PGF1α, and LTB4) significantly induced hemocyte nodulation and phagocytosis in the presence of dexamethasone, an eicosanoid biosynthesis inhibitor. However, only PGs induced cell lysis of oenocytoids to release prophenoloxidase, which resulted in an increase in phenoloxidase activity. These seven eicosanoids also induced expression of humoral immune-associated genes, including prophenoloxidase, serpin, dopa decarboxylase, cecropin, and lysozyme, in which PGB2 and PGE1 did not induce gene expression of prophenoloxidase. To understand the interactions between different eicosanoids, mixture effects of these eicosanoids were compared with their individual eicosanoid effects on mediating nodule formation in response to bacterial challenge. All six single PGs showed increases in nodule formation in a dose-dependent manner without significant difference among the different types. LTB4 was more potent than the tested PGs in mediating the cellular immune response. At low doses, all combinations of two eicosanoids showed significant additive effects on nodule formation. These results indicate that immune target cells, such as hemocyte and fat body, of S. exigua can respond to different COX and LOX products to express cellular and humoral immune responses, and their overlapping, additive effects on nodulation suggest that in target cells, these eicosanoids share a hypothetical common eicosanoid signal pathway.

Genes encoding phospholipases A2 mediate insect nodulation reactions to bacterial challenge.

We propose that expression of four genes encoding secretory phospholipases A(2) (sPLA(2)) mediates insect nodulation responses to bacterial infection. Nodulation is the quantitatively predominant cellular defense reaction to bacterial infection. This reaction is mediated by eicosanoids, the biosynthesis of which depends on PLA(2)-catalyzed hydrolysis of arachidonic acid (AA) from cellular phospholipids. Injecting late instar larvae of the red flour beetle, Tribolium castaneum, with the bacterium, Escherichia coli, stimulated nodulation reactions and sPLA(2) activity in time- and dose-related manners. Nodulation was inhibited by pharmaceutical inhibitors of enzymes involved in eicosanoid biosynthesis, and the inhibition was rescued by AA. We cloned five genes encoding sPLA(2) and expressed them in E. coli cells to demonstrate these genes encode catalytically active sPLA(2)s. The recombinant sPLA(2)s were inhibited by sPLA(2) inhibitors. Injecting larvae with double-stranded RNAs specific to each of the five genes led to reduced expression of the corresponding sPLA(2) genes and to reduced nodulation reactions to bacterial infections for four of the five genes. The reduced nodulation was rescued by AA, indicating that expression of four genes encoding sPLA(2)s mediates nodulation reactions. A polyclonal antibody that reacted with all five sPLA(2)s showed the presence of the sPLA(2) enzymes in hemocytes and revealed that the enzymes were more closely associated with hemocyte plasma membranes following infection. Identifying specific sPLA(2) genes that mediate nodulation reactions strongly supports our hypothesis that sPLA(2)s are central enzymes in insect cellular immune reactions.

Biochemical characteristics of immune-associated phospholipase A2 and its inhibition by an entomopathogenic bacterium, Xenorhabdus nematophila

An entomopathogenic bacterium, Xenorhabdus nematophila, induces an immunosuppression of target insects by inhibiting phospholipase A2 (PLA2) activity. Recently, an immune-associated PLA2 gene was identified from the red flour beetle, Tribolium castaneum. This study cloned this PLA2 gene in a bacterial expression vector to produce a recombinant enzyme. The recombinant T. castaneum PLA2 (TcPLA2) exhibited its characteristic enzyme activity with substrate concentration, pH, and ambient temperature. Its biochemical characteristics matched to a secretory type of PLA2 (sPLA2) because its activity was inhibited by dithiothreitol (a reducing agent of disulfide bond) and bromophenacyl bromide (a specific sPLA2 inhibitor) but not by methylarachidonyl fluorophosphonate (a specific cytosolic type of PLA2). The X. nematophila culture broth contained PLA2 inhibitory factor(s), which was most abundant in the media obtained at a stationary bacterial growth phase. The PLA2 inhibitory factor(s) was heat-resistant and extracted in both aqueous and organic fractions. Effect of a PLA2-inhibitory fraction on the immunosuppression of T. castaneum was equally comparable with that resulted from inhibition of the TcPLA2 gene expression by RNA interference.

Activation of immune-associated phospholipase A2 is functionally linked to Toll/Imd signal pathways in the red flour beetle, Tribolium castaneum

Bacterial challenge enhances phospholipase A(2) (PLA(2)) activity, which in turn induces biosynthesis of various eicosanoids that mediate non-self recognition signal to immune effectors in insects. But, there is little information on how PLA(2) activity is controlled after the non-self recognition. A recent genome analysis of the red flour beetle, Tribolium castaneum, has annotated both Toll and Imd signal pathways that are presumably considered to specifically respond to different microbial infections to express specific antimicrobial peptides (AMPs). This study set up a hypothesis that PLA(2) activation is linked to Toll and Imd pathways in T. castaneum. Bacterial challenge to the larvae of T. castaneum induced expressions of Toll and Imd genes. Different AMP genes were induced in larvae infected with Gram-positive or -negative bacteria. RNA interference using double-stranded RNAs (dsRNAs) specific to different Toll and Imd genes showed differential inhibition of these AMP expressions, indicating that the Toll and Imd pathways play critical roles in the production of AMPs by specifically responding to various bacterial challenges in T. castaneum. These Toll and Imd immune signals are also linked to the activation of PLA(2) in T. castaneum. Activation of PLA(2) was significantly induced in response to bacterial challenge, but was inhibited by dsRNAs specific to different Toll and Imd genes. The activation of PLA(2) via Toll and Imd pathways could be explained by induction of PLA(2) gene expression because the dsRNA treatments of Toll and Imd genes suppressed the gene expression of PLA(2) in response to bacterial challenge. The functional links were further supported by an immunofluorescence assay of PLA(2) intracellular translocation. Upon bacterial challenge, hemocytes from control larvae showed intracellular translocation of their PLA(2)s near to cell membrane, but hemocytes from larvae treated with dsRNAs of the Toll and Imd genes did not show the translocation, at which the PLA(2)s appeared to be evenly spread in the cytoplasm. These results suggest that recognition of bacterial challenge initiates Toll and Imd pathways in T. castaneum, which subsequently induces the activation of immune-associated PLA(2)s as well as gene expression of various AMPs.

Role of a small G protein Ras in cellular immune response of the beet armyworm, Spodoptera exigua.

Insect cellular immune responses accompany cytoskeletal rearrangement of hemocytes to exhibit filopodial and pseudopodial extension of their cytoplasm. Small G proteins are postulated to be implicated in the hemocyte cellular processes to perform phagocytosis, nodulation, and encapsulation behaviors. A small G protein ras gene (Se-Ras) was cloned from cDNAs prepared from hemocytes of the beet armyworm, Spodoptera exigua. The open reading frame of Se-Ras encoded 179 amino acids with a predicted molecular weight of 20.0kDa, in which 114 residues at amino terminus were predicted to be a GTP binding domain. It showed high sequence similarities (86.1-92.8%) with known ras genes in other insects. Se-Ras was constitutively expressed in all developmental stages from egg to adult without any significant change in expression levels in response to bacterial challenge. A specific double strand RNA (dsRNA) could knockdown its expression in the hemocytes after 48h post-injection. While the RNA interference (RNAi) did not show any change in total or differential hemocyte counts, it impaired hemocyte behaviors. The RNAi of Se-Ras significantly suppressed hemocyte spreading, cytoskeleton extension, and nodulation behaviors in response to bacterial challenge. Release of prophenoloxidase from oenocytoids was significantly inhibited by the RNAi, which resulted in significant suppression in PO activation in response to an inducer, PGE(2). These results suggest that Se-Ras is implicated in mediating cellular processes of S. exigua hemocytes. This is the first report of Ras role in insect cellular immune response.

Factors Affecting the Activation of Hemolymph Prophenoloxidase of Spodoptera exigua (Lepidoptera: Noctuidae)

Abstract In insect immunity, phenoloxidase (PO) plays an important role in the processes during melanotic encapsulation and phagocytosis. However, uncontrolled PO activity is known to be fatal due to its toxic catalytic products. Thus, the activation of prophenoloxidase (proPO) to PO must be controlled. This study showed that at least three factors were involved in the proPO activation pathway of Spodoptera exigua . Most hemolymph PO activity of S. exigua was detected in hemocytes. The de novo synthesis of proPO was a factor by which PO activity levels were determined in hemolymph, as PO activity was inhibited in a dose-dependent manner by cycloheximide, a eukaryotic protein translation inhibitor. The second factor was derived from serine protease(s) becasue only serine protease inhibitors prevented proPO activation, while other proteases did not. In addition, eicosanoids were implicated in proPO activation, because dexamethasone, an inhibitor of phospholipase A 2 , inhibited proPO activation. These results indicate that the activation of proPO in S. exigua is controlled at both transcriptional and posttranscriptional levels.

An immunological role of a PKC alpha binding protein (PICK1) expressed in the hemocytes of the beet armyworm, Spodoptera exigua

Protein kinase C (PKC) regulates various intracellular processes and its activity is tightly controlled by various factors, such as secondary messengers and binding proteins. A cDNA of a PKC alpha binding protein (also called PICK1: protein interacting with C kinase 1) was cloned in hemocytes of the beet armyworm, Spodoptera exigua (Noctuidae: Lepidoptera). It encodes 475 amino acid residues with putative PDZ and BAR domains interacting with other proteins or ligands. The PICK1 gene of S. exigua (Se-PICK1) was expressed in all developmental stages. In the larval stage, it was highly expressed in hemocyte and brain tissues. A quantitative RT-PCR indicated that its expression was significantly up-regulated by a bacterial challenge. RNA interference of Se-PICK1 in the fifth instar larvae with 100ng of a specific double-stranded RNA could effectively knockdown its expression after 48h post-injection in hemocytes. The suppressed expression of Se-PICK1 significantly impaired the larvae of S. exigua to induce hemocyte-spreading behavior and to form hemocyte nodules in response to bacterial infection. This is the first report of an immunological role of PICK1, which has been identified in various insect and mammalian genomes.