Jon S. Miller

Eicosanoid actions in insect cellular immune functions

Insects are more or less constantly challenged with a daunting array of pathogenic organisms, including viruses, bacteria, fungi, protozoans as well as various metazoan parasites and parasitoids. At the first level of defense, the pathogens are rebuffed by physical barriers, including the cuticle and peritrophic membrane. Upon breaching these barriers, pathogens meet with an arsenal of robust and efficacious immune defense mechanisms. Two general categories of defenses are typically recognized, humoral defenses and hemocytic or cellular defenses. The former involves induced synthesis of various antibacterial proteins and peptides, such as cecropins and lysozyme. Cellular defense mechanisms are characterized by direct interactions between circulating hemocytes and the invaders. These include phagocytosis, microaggregation, nodulation, and encapsulation. Microaggregation is a step in the nodulation process, which is responsible for clearing the bulk of bacterial infections from circulation. Coordinated cellular actions lead to encapsulation of invaders, such as parasitoid eggs, that are very much larger than individual hemocytes. While the defense mechanisms are broadly appreciated, less is known about the biochemical signals responsible for mediating and coordinating the cellular actions. We now know eicosanoids mediate phagocytosis, microaggregation, and nodulation reactions to immune challenge, as well as cell spreading, a specific step in nodulation. We have several goals in this mini review. We provide a brief background on cellular immunity, outline eicosanoid biosynthesis, and review eicosanoid actions in cellular immunity in insects. Recent work indicates some pathogens have usurped eicosanoid‐mediated immunity; they disable insect immunity by inhibiting eicosanoid biosynthesis. We interpret these findings and their significance with respect to the biological control of insects. We also present preliminary work designed to test hypotheses on how eicosanoids exert their actions. We address shortcomings in our knowledge on eicosanoids in insect biology.

Eicosanoid Actions in Insect Immunity

Insects express 3 lines of protection from infections and invasions. Their cuticles and peritrophic membranes are physical barriers. Infections and invasions are quickly recognized within insect bodies, and recognition launches 2 lines of innate immune reactions. Humoral reactions involve induced synthesis of antimicrobial peptides, the bacteriolytic enzyme lysozyme and activation of the prophenoloxidase system. Cellular immune reactions include phagocytosis, nodulation and encapsulation. These reactions entail direct interactions between circulating hemocytes and the invaders. Cellular immune reactions begin immediately after an invasion is detected while antimicrobial peptides typically appear in the hemolymph some hours after infection. Microaggregation is a step in the nodulation process, which is responsible for clearing the bulk of bacterial infections from circulation. Coordinated cellular actions lead to encapsulation of invaders, such as parasitoid eggs, that are very much larger than individual hemocytes. In this paper, we review the roles of eicosanoids as central mediators of insect immune reactions, particularly cellular reactions. We briefly describe insect immune functions, outline eicosanoid biosynthesis and treat eicosanoid actions in cellular immunity of insects. Eicosanoids act in several cellular defense functions, including phagocytosis, microaggregation, nodulation, encapsulation, cell spreading and hemocyte migration toward a source of a bacterial peptide. We also describe our most recent work on the influence of one group of eicosanoids, prostaglandins, on gene expression in an established insect cell line.

Eicosanoids mediate nodulation responses to bacterial infections in larvae of the tenebrionid beetle, Zophobas atratus

Nodulation is the temporally and quantitatively predominant cellular defense response to bacterial infection in insects and other invertebrates. Inhibition of eicosanoid biosynthesis in larvae of the tenebrionid beetle, Zophobas atratus, immediately prior to intrahemocoelic injections with heat killed preparations of the bacterium, Serratia marcescens, strongly reduced the nodulation response. Separate treatments with specific inhibitors of phospholipase A2, cyclooxygenase, and lipoxygenase reduced nodulation, supporting the view that nodule formation is a complex process involving both cyclooxygenase and lipoxygenase products. The inhibitory effects of the phospholipase A2 inhibitor, dexamethasone, on nodulation were apparent 1 h after infection, and the effects increased, relative to controls, over 24 h. The dexamethasone effects were expressed in a dose-dependent manner, and they were reversed by treating bacteria injected insects with the eicosanoid-precursor polyunsaturated fatty acid, arachidonic acid (C20:4n-6). Treatments with the saturated fatty acid, 16:0, which is not an eicosanoid precursor, did not reverse the dexamethasone effects on nodulation. The insects contain low levels of three eicosanoid precursor polyunsaturated fatty acids in six different tissues, and fat body preparations are competent to produce both cyclooxygenase and lipoxygenase products. These findings strongly support the identification of nodulation as a specific insect cellular defense mechanism that is mediated by eicosanoids.

Eicosanoids mediate microaggregation and nodulation responses to bacterial infections in black cutworms, Agrotis ipsilon, and true armyworms, Pseudaletia unipuncta

Nodulation is the first, and quantitatively predominant, cellular defense reaction to bacterial infection in insects and other invertebrates. Inhibition of eicosanoid biosynthesis in true armyworms, Pseudaletia unipuncta, and black cutworms, Agrotis ipsilon, immediately prior to intrahemocoelic injections with heat-killed preparations of the bacterium, Serratia marcescens, severely impaired the nodulation response. Five eicosanoid biosynthesis inhibitors, including dexamethasone (a phospholipase A(2) inhibitor), indomethacin, ibuprofen (cyclooxygenase inhibitors), phenidone (dual lipoxygenase/cyclooxygenase inhibitor) and eicosatetraynoic acid (an arachidonic acid analog that inhibits all arachidonic acid metabolism) severely reduced nodulation in infected insects. The dexamethasone effects were reversed by treating true armyworms with arachidonic acid immediately after infection. In addition to these pharmacological findings, we demonstrate that an eicosanoid biosynthesis system is present in these insects. Arachidonic acid is present in fat body phospholipids at about 0.4% of total phospholipid fatty acids. Fat body expressed a phospholipase A(2) that can hydrolyze arachidonic acid from the sn-2 position of cellular phospholipids. Fat body preparations were competent to biosynthesize prostaglandins, of which PGE(2) was the major product. These findings support the hypothesis that eicosanoids mediate cellular immune reactions in insects.

Eicosanoids mediate nodulation reactions to bacterial infections in adults of the cricket, Gryllus assimilis

Nodulation is the temporally and quantitatively most important cellular defense reaction to bacterial infections in insects. Inhibition of eicosanoid biosynthesis in adults of the cricket, Gryllus assimilis, immediately prior to intrahemocoelic injections of the bacterium, Serratia marcescens, sharply reduced the nodulation response. Separate treatments with specific inhibitors of phospholipase A(2), cyclooxygenase, and lipoxygenase reduced nodulation, supporting our view that nodule formation is a complex process involving lipoxygenase and cyclooxygenase products. The inhibitory influence of dexamethasone was apparent within 2h of injection, and nodulation was significantly reduced, relative to control crickets, over 22h. The dexamethasone effects were reversed by treating bacteria-injected insects with the eicosanoid-precursor polyunsaturated fatty acid, arachidonic acid. Low levels of arachidonic acid were detected in fat body phospholipids, and fat body preparations were shown to be competent to biosynthesize eicosanoids from exogenous radioactive arachidonic acid. These findings in a hemimetabolous insect broaden our hypothesis that eicosanoids mediate cellular immune reactions to bacterial infections in most, if not all, insects.

Eicosanoids mediate microaggregation reactions to bacterial challenge in isolated insect hemocyte preparations

Nodule formation is the quantitatively predominant insect cellular defense reaction to bacterial challenges, responsible for clearing the largest proportion of infecting bacteria from circulation. It has been suggested that eicosanoids mediate several steps in the nodulation process, including formation of hemocyte microaggregates, an early step in the process. While fat body and hemocytes are competent to biosynthesize eicosanoids, the source of the nodulation-mediating eicosanoids remains unclear. To investigate this issue, we studied hemocyte microaggregation reactions to bacterial challenge in vitro. Hemocyte suspensions from the tobacco hornworm, Manduca sexta, were treated with the phospholipase A(2) inhibitor, dexamethasone, then challenged with the bacterium Serratia marcescens. Preparations treated with dexamethasone yielded fewer hemocyte microaggregations than untreated, control preparations. Furthermore, the influence of dexamethasone was reversed by amending experimental (dexamethasone-treated) preparations with the eicosanoid biosynthesis precursor, arachidonic acid. Palmitic acid, which is not a substrate for eicosanoid biosynthesis, did not reverse the influence of dexamethasone on the microaggregation reaction. The influence of dexamethasone was also reversed by adding filtered media from challenged hemocyte preparations to dexamethasone-treated preparations. Finally, most hemocyte preparations treated with selected eicosanoid biosynthesis inhibitors formed fewer hemocyte microaggregations than control preparations. The 5- and 12-lipoxygenase inhibitor, esculetin, did not influence the formation of hemocyte microaggregations in this system. These results are consistent with similar investigations performed in vivo, and we infer that hemocytes are responsible for forming and secreting eicosanoids, which subsequently initiate nodulation by mediating hemocyte microaggregation.

The influence of bacterial species and intensity of infections on nodule formation in insects

Nodulation is the predominant cellular immune reaction to bacterial infection in insects. Nodulation is a complex process involving an unknown number of discrete cellular actions. Currently, there is only limited information on the signal transduction mechanisms that result in nodulation. In older larvae of the tobacco hornworm, Manduca sexta, and of the tenebrionid beetle, Zophobas atratus, eicosanoids are involved in one or more steps in the overall process, and treating these insects with inhibitors of eicosanoid biosynthesis prior to bacterial infection severely impairs their ability to form nodules. In this paper we address more detailed questions on eicosanoid-mediated nodulation. The nodulation reaction to bacterial infection occurs in all larval stages we examined, specifically, second, third, and fourth instars of M. sexta. In both species, the number of nodules formed in response to bacterial infection is related in an exponential way to the number of bacterial cells in the infection. Nodulation is also not related to larval size. We also found that nodulation intensity varies according to the species of infecting bacteria.

Eicosanoids mediate nodulation reactions to bacterial infections in adults of two 17-year periodical cicadas, Magicicada septendecim and M. cassini.

Nodulation is the first and quantitatively most important cellular defense reaction to bacterial infections in insects. Treating adults of the 17-year periodical cicadas, Magicicada septendecim and M. cassini, with eicosanoid biosynthesis inhibitors immediately prior to intrahemocoelic injections of the bacterium, Serratia marcescens, sharply reduced the nodulation response to bacterial challenges. Separate treatments with specific inhibitors of phospholipase A(2), cyclooxygenase, and lipoxygenase reduced nodulation, supporting our view that nodule formation is a multi-step process in which individual steps are separately mediated by lipoxygenase and cyclooxygenase products. The inhibitory influence of dexamethasone was apparent by 2 h after injection, and nodulation was significantly reduced, relative to control insects, over the following 14 h. The dexamethasone effects were reversed by treating bacteria-challenged insects with the eicosanoid-precursor polyunsaturated fatty acid, arachidonic acid. Low levels of arachidonic acid were detected in fat body phospholipids. These findings in adults of an exopterygote insect species with an unusual life history pattern broaden our hypothesis that eicosanoids mediate cellular immune reactions to bacterial infections in most, if not all, insects.

Eicosanoids Mediate Nodulation Responses to Bacterial Infections in Larvae of the Silkmoth, Bombyx mori

Abstract 1) Nodulation is the first, and qualitatively predominant, cellular defense reaction to bacterial infections in insects and other invertebrates; 2) treating silkworms, Bombyx mori, with the eicosanoid biosynthesis inhibitor, dexamethasone, strongly reduced nodulation responses to bacterial infections; 3) the influence of dexamethasone was reversed by injecting the eicosanoid-precursor polyunsaturated fatty acid, arachidonic acid (20:4n-6), into dexamethasone-treated, infected larvae; 4) the presence of an eicosanoid biosynthesis system in silkworms was documented. Demonstrated elements include a digestive phospholipase A2, incorporation of exogenous 20:4n-6 into fat body phospholipids, the presence of 20:4n-6 in cellular phospholipids, a fat body intracellular phospholipase A2 that can hydrolyze 20:4n-6 from cellular phospolipids, and eicosanoid biosynthetic enzymes; and 5) these findings support the hypothesis that eicosanoids mediate cellular immune responses to bacterial infections in silkworms.

A DIGESTIVE PHOSPHOLIPASE A2 IN THE TIGER BEETLE CICINDELLA CIRCUMPICTA

Abstract We report on phospholipase A 2 (PLA 2 ) in the midgut contents of tiger beetles Cicindella circumpicta . PLA 2 is responsible for hydrolyzing fatty acids from the sn -2 position of dietary phospholipids (PLs), an essential step in the digestion and absorption of essential fatty acids. As expected from the background of mammalian digestive PLA 2 s, the midgut PLA 2 was Ca 2+ -dependent. The PLA 2 in tiger beetle midgut contents was influenced by altering the reaction conditions, including incubation time, protein concentrations, pH and temperature. Routine assay conditions included incubating 3.3 μg of protein prepared from midgut contents in Tris buffer (pH 9.0) for 30 min at 28 °C. Partial purification on size-exclusion chromatography indicated that the molecular weight of the midgut PLA 2 is approximately 22 kDa. The partially purified PLA 2 was inhibited by the site-specific inhibitor oleyoxyethyl phosphorylcholine. The presence of the PLA 2 in the midgut contents is related to feeding activity, because the midgut contents of unfed beetles did not express PLA 2 . We suggest that PLA 2 s are a common, and quite important, feature of insect digestive physiology.