Eicosanoids mediate Galleria mellonella cellular immune response to viral infection

Nodulation is the predominant insect cellular immune response to bacterial and fungal infections and it can also be induced by some viral infections. Treating seventh instar larvae of greater wax moth Galleria mellonella with Bovine herpes simplex virus-1 (BHSV-1) induced nodulation reactions in a dose-dependent manner. Because eicosanoids mediate nodulation reactions to bacterial and fungal infection, we hypothesized that eicosanoids also mediate nodulation reactions to viral challenge. To test this idea, we injected G. mellonella larvae with indomethacin, a nonsteroidal anti-inflammatory drug immediately prior to intrahemocoelic injection of BHSV-1. Relative to vehicle-treated controls, indomethacin-treated larvae produced significantly reduced numbers of nodules following viral infection (down from approximately 190 nodules/larva to <50 nodules/larva). In addition to injection treatments, increasing dietary indomethacin dosages (from 0.01% to 1%) were associated with decreasing nodulation (by 10-fold) and phenoloxidase activity (by 3-fold) reactions to BHSV-1 injection. We infer from these findings that cyclooxygenase products, prostaglandins, mediate nodulation response to viral infection in G. mellonella.     

Eicosanoids mediate nodulation reactions to a mollicute bacterium in larvae of the blowfly, Chrysomya megacephala

Nodulation is the temporally and quantitatively most important cellular defense response to bacterial, fungal and some viral infections in insects. We tested the hypothesis that prostaglandins and other eicosanoids are responsible for mediating nodulation reactions to bacterial infection in larvae of the blowfly Chrysomya megacephala. Third-instar larvae treated with Ureaplasma urealyticum formed nodules in a challenge dose-dependent manner. Nodulation was evoked shortly after injection and reached a maximum of approximately 25 nodules/larva within 8 h. Larvae treated with the glucocorticoid, dexamethasone and the cyclooxygenase inhibitors, indomethacin and piroxicam were impaired in their ability to form nodules following U. urealyticum infection. The number of nodules decreased with increasing doses of piroxicam. Contrarily, treating larvae with the lipooxygenase inhibitor, esculetin, and the dual cyclooxygenase/lipooxygenase inhibitor, phenidone did not influence nodulation reactions to infection. Supplying dexamethasone-treated larvae with the eicosanoid precursor, arachidonic acid, reversed the inhibitory effect of dexamethasone on nodulation. We infer from these results that eicosanoids mediate nodulation reactions to infection of a bacterial species that lacks cell walls in larvae of the blowfly, C. megacephala.     

EICOSANOIDS MEDIATE CELLULAR IMMUNE RESPONSE AND PHENOLOXIDASE REACTION TO VIRAL INFECTION IN ADULT Pimpla turionellae

Nodulation is the predominant insect cellular immune response to microbial infections. We posed the hypothesis that parasitoid insects in their adulthood express melanotic nodulation reactions to viral challenge and that eicosanoids mediate nodulation reactions and phenoloxidase (PO) activation in response to viral challenge. To test this idea, we injected Pimpla turionellae adults with indomethacin, a nonsteroidal anti-inflammatory drug, immediately prior to intrahemocoelic injection of Bovine herpes simplex virus-1 (BHSV-1). Treating newly emerged adults of P. turionellae with BHSV-1 induced nodulation reactions, and decreased PO activity at high viral doses. Relative to vehicle-treated controls, indomethacin-treated adults produced significantly reduced numbers of nodules following viral infection (down from approximately 21 nodules per adult to less than six nodules per adult). In addition to injection treatments, increasing dietary indomethacin dosages (from 0.01% to 0.1%) were associated with decreasing nodulation (by six-fold) and PO (by about three-fold) reactions to BHSV-1 injection. Wasp adults orally fed with the lowest dietary indomethacin concentration (0.001%) expressed significantly increased PO activity (1.45 unit/min/mg protein) while nodulation reaction was not affected in response to viral challenge compared to control adults. We infer from these findings that cyclooxygenase (COX) products, at least prostaglandins, mediate nodulation response and PO action to viral infection in adults of these highly specialized insects.     

Eicosanoids act in nodulation reactions to bacterial infections in newly emerged adult honey bees, Apis mellifera, but not in older foragers

Nodulation is the first, and qualitatively predominant, cellular defense reaction to bacterial infections in insects. We tested the hypothesis that eicosanoids also mediate nodulation reactions to bacterial challenge in adults of a social insect, the honey bee, Apis mellifera. Treating newly-emerged experimental bees with the eicosanoid biosynthesis inhibitor, dexamethasone, impaired nodulation reactions to bacterial infections, and the influence of dexamethasone was reversed by treating infected insects with arachidonic acid, an eicosanoid precursor. Several other eicosanoid biosynthesis inhibitors, including the cyclooxygenase inhibitor, indomethacin, and the dual cyclooxygenase/lipoxygenase inhibitor, phenidone, also impaired the ability of experimental honeybees to form nodules in reaction to bacterial challenge. The influence of phenidone on nodulation was expressed in a dose-dependent manner. However, in experiments with older honey bees foragers, similar bacterial challenge did not evoke nodulation reactions. We infer from our results that while eicosanoids mediate cellular immune responses to bacterial infections in newly emerged honey bees, and more broadly, in most insect species, nodulation reactions to bacterial challenge probably do not occur in all phases of insect life cycles.     

Prostaglandins, not lipoxygenase products, mediate insect microaggregation reactions to bacterial challenge in isolated hemocyte preparations

Nodulation is the predominant cellular defense reaction to bacterial challenge in insects. Eicosanoids mediate several steps in the nodulation process, including formation of hemocyte microaggregations. Isolated hemocyte preparations synthesize and secrete eicosanoids, which mediate hemocytic immune reactions. Two major groups of eicosanoids are prostaglandins (products of cyclooxygenase pathways) and various products of lipoxygenase pathways. In this study, we test the hypothesis that prostaglandins, but not lipoxygenase products, mediate hemocyte microaggregation reactions in response to bacterial challenge. Our results indicate that isolated hemocyte preparations pretreated with the cyclooxygenase inhibitors indomethacin and naproxen yielded fewer microaggregates than untreated control groups (3.7 x 10(5) microaggregates/ml hemolymph vs. 11.0 x 10(5) microaggregates/ml hemolymph). These inhibitors influence hemocyte microaggregate formation in a dose-dependent manner in treatments ranging from 0 to 200 microM. The lipoxygenase inhibitors esculetin and caffeic acid did not impact the formation of microaggregates in this system. The influence of the phospholipase A(2) inhibitor dexamethasone was reversed by amending experimental (dexamethasone-treated) preparations with prostaglandin H(2), but not prostaglandin D(2), prostaglandin E(2), nor 5(S)-hydroperoxy-6E,8Z,11Z,14Z-eicosatetraenoic acid, a product of the lipoxygenase pathway. We infer that prostaglandins are the primary mediators of microaggregation reactions to bacterial challenge in insect hemocyte preparations.     

Eicosanoids mediate the laminarin-induced nodulation response in larvae of the flesh fly, Neobellieria bullata

Insects have a highly developed innate immune system, including humoral and cellular components. The cellular immune responses refer to hemocyte-mediated processes such as phagocytosis, nodulation, and encapsulation. Nodulation is considered the predominant defense reaction to infection in insects. Treating third instar larvae of the grey flesh fly, Neobellieria bullata, with laminarin (beta-1,3-glucan, a typical component of fungal cell walls) induced nodulation in a dose-dependent manner. This reaction was initiated very soon after injection and reached its maximal response level after 4 h. The nodules were not randomly distributed in the hemocoel, but were concentrated around the crop. The possible role of eicosanoids in this nodulation process was determined by treating larvae with the phospholipase A(2) inhibitor, dexamethasone, the cyclooxygenase inhibitor, naproxen, and the lipoxygenase inhibitor, esculetin. Both dexamethasone and naproxen significantly impaired the ability of N. bullata larvae to form nodules in response to laminarin. Supplying dexamethasone-treated larvae with the eicosanoid precursor, arachidonic acid, restored the full response. On the other hand, treating larvae with esculetin did not influence the formation of nodules in response to laminarin. This is the first study that demonstrates the occurrence of a laminarin-induced nodulation response in Diptera. Phospholipase A(2) and cyclooxygenase activities, both involved in prostaglandin biosynthesis, appear to play an important role in the regulation of this process.     

Insect cellular reactions to the lipopolysaccharide component of the bacterium Serratia marcescens are mediated by eicosanoids

Nodulation, which begins with the formation of cellular microaggregates, is the predominant cellular defense reaction to bacterial infections in insects. We suggested that these reactions to bacterial infections are mediated by eicosanoids. The lipopolysaccharide (LPS) component of some bacterial cells stimulates defense reactions in mammals and insects. Here, we report on experiments designed to test the hypothesis that eicosanoids mediate microaggregation reactions to LPS. Injections of LPS (purified from the bacterium, Serratia marcescens) into larvae of the tenebrionid beetle, Zophobas atratus, stimulated microaggregation reactions in a dose-dependent manner. Treatments with eicosanoid-biosynthesis inhibitors immediately prior to LPS challenge sharply reduced the microaggregation responses. Separate treatments with specific inhibitors of phospholipase A(2), cyclooxygenase and lipoxygenase reduced microaggregation, supporting our view that microaggregate formation involves lipoxygenase and cyclooxygenase products. The inhibitory influence of dexamethasone was apparent within 30min after injection, and microaggregation was significantly reduced, relative to control insects, over the following 90min. The dexamethasone effects were reversed by treating LPS-injected insects with the eicosanoid precursor, arachidonic acid. These findings indicate that cellular defense reactions to a specific component of bacterial cells are mediated by eicosanoids, and open up new possibilities for dissecting detailed hemocytic actions in insect immune reactions to bacterial infections.     

Eicosanoid biosynthesis inhibitors influence mortality of Pieris brassicae larvae co-injected with fungal conidia

Influence of fungal species (conidia spores) on mortality of Pieris brassicae larvae differed when injected into the larvae. The effects of B. bassiana (ARSEF-1151) were expressed in a conidial dose-dependent manner on mortality of the larvae. An increased and faster mortality of the larvae followed B. bassiana (ARSEF-1151) infection when the spores were co-injected with the eicosanoid biosynthesis inhibitors (dexamethasone, naproxen, phenidone, esculetin). These compounds express different modes of action. These lethal effects were reversed when dexamethasone was injected with eicosanoid precursor arachidonic acid (20:4n-6). Nodulation is the predominant cellular reaction to bacterial and fungal injection in insects. Injection of each of five fungal species showed that nodulation intensity varies according to infecting fungal species. These findings support the idea that virulent effects of entomopathogenic, fungal species can be increased when P. brassicae immune systems are suppressed.     

Eicosanoids mediate nodulation reactions to bacterial Escherichia coli K 12 infections in larvae of the oriental blowfly, Chrysomya megacephala

Abstract  Nodulation is the predominant cellular defense reaction to bacterial challenges in insects. In this study, third instar larvae of Chrysomya megacephala were injected with bacteria, Escherichia coli K 12 (10⁶ CFU/mL, 2 μL), immediately prior to injection of inhibitors of eicosanoid biosynthesis, which sharply reduced nodulation response. Test larvae were treated with specific inhibitors of phospholipase A₂ (dexamethasone), cyclo-oxygenase (indomethacin, ibuprofen and piroxicam), dual cyclo-oxygenase/lipoxygenase (phenidone) and lipoxygenase (esculetin) and these reduced nodulation except esculetin. The influence of bacteria was obvious within 2 h of injection (5 nodules/larva), and increased to a maximum after 8 h (with 15 nodules/larva), and then significantly reduced over 24 h (9 nodules/larva). The inhibitory influence of dexamethasone was apparent within 2 h of injection (4 vs. 5 nodules/larva), and nodulation was significantly reduced, compared to control, over 24 h (5 vs. 8 nodules/larva). Increased dosages of ibuprofen, indomethacin, piroxicam and phenidone led to decreased numbers of nodules. Nodules continued to exist during the pupal stage. However, the effects of dexamethasone were reversed by treating bacteria-injected insects with an eicosanoid-precursor polyunsaturated fatty acid, arachidonic acid. These findings approved our view that eicosanoid can mediate cellular defense mechanisms in response to bacterial infections in another Dipteran insect C. megacephala .     

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.     

Eicosanoids influence in vitro elongation of plasmatocytes from the tobacco hornworm, Manduca sexta

Nodule formation is the predominant insect cellular defense reaction to bacterial challenges, responsible for clearing the largest proportion of infecting bacteria from hemolymph circulation. Hemocyte spreading behavior is a critical step in the nodulation process. It has been suggested that eicosanoids mediate several steps in the process. However, the influence of eicosanoids on hemocyte spreading has not been investigated in detail. To test the hypothesis that eicosanoids mediate hemocyte spreading behavior, I treated larvae of the tobacco hornworm, Manduca sexta, with eicosanoid biosynthesis inhibitors and later assessed plasmatocyte elongation on glass slides. Plasmatocytes from larvae treated with dexamethasone did not elongate to the extent of plasmatocytes from untreated control larvae. The dexamethasone effect on plasmatocyte elongation was expressed in a dose-dependent manner and was reversed by injecting dexamethasone-treated larvae with the eicosanoid-precursor fatty acid, arachidonic acid. Palmitic acid, which is not substrate for eicosanoid biosynthesis, did not reverse the influence of dexamethasone on plasmatocyte elongation. Finally, plasmatocytes from larvae treated with a range of eicosanoid biosynthesis inhibitors did not elongate to the extent of plasmatocytes from control larvae. Plasmatocyte width did not appear to be influenced in this study. These findings strongly support the idea that insect plasmatocyte elongation is influenced 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.     

The Effects of Eicosanoid Biosynthesis Inhibitors On Prophenoloxidase Activation,Phagocytosis and Cell Spreading in Galleria mellonella

The invertebrate immune system produces melanotic nodules in response to bacterial infections and this has previously been shown to be mediated by eicosanoids. Nodulation occurs in two phases: the first involves hemocyte degranulation and activation of the prophenoloxidase cascade; the second involves formation of a cellular capsule by attachment and spreading of hemocytes. We demonstrate that inhibitors of eicosanoid biosynthesis affect both of these phases of nodulation in Galleria mellonella. The phospholipase A(2) inhibitor, dexamethasone, as well as the cyclooxygenase inhibitor, indomethacin, significantly inhibit phagocytosis in vitro and prophenoloxidase activation in vivo. The inhibitory effects of dexamethasone were abolished by the addition of exogenous arachidonic acid. Furthermore, 5,8,11,14- eicosatetraynoic acid, dexamethasone and indomethacin inhibit hemocyte spreading in vitro. The findings support the idea that eicosanoid derivatives mediate both phases of the nodulation response and are consistent with previous studies which attribute roles for eicosanoids in other species as modulators of cell activity.     

Prostaglandin‐mediated recovery from bacteremia delays larval development in fall armyworm,Spodoptera frugiperda

Insect immunity includes a surveillance system that detects and signals infections, coupled with hemocytic and humoral immune functions. These functions are signaled and coordinated by several biochemicals, including biogenic amines, insect cytokines, peptides, and prostaglandins (PGs). The actions of these mediators are coordinated within cells by various forms of cross‐talk among the signaling systems and they result in effective reactions to infection. While this is well understood, we lack information on how immune‐mediated recovery influences subsequent juvenile development in surviving insects. We investigated this point by posing the hypothesis that PG signaling is necessary for larval recovery, although the recovery imposes biological costs, registered in developmental delays and failures in surviving individuals. Here, we report that nodulation responses to infections by the bacterium, Serratia marcescens, increased over time up to 5 h postinfection, with no further nodulation; it increased in a linear manner with increasing bacterial dosages. Larval survivorship decreased with increasing bacterial doses. Treating larvae with the PG‐biosynthesis inhibitor, indomethacin, led to sharply decreased nodulation reactions to infection, which were rescued in larvae cotreated with indomethacin and the PG‐precursor, arachidonic acid. Although nodulation was fully rescued, all bacterial challenged larvae suffered reduced survivorship compared to controls. Bacterial infection led to reduced developmental rates in larvae, but not pupae. Adult emergence from pupae that developed from experimental larvae was also decreased. Taken together, our data potently bolster our hypothesis.     

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 Manduca sexta cellular response to the fungal pathogen Beauveria bassiana: a role for the lipoxygenase pathway

Many studies have documented the involvement of eicosanoids in insect cellular immune responses to bacteria. The use of the fungal pathogen Beauveria bassiana as a nodulation elicitor, with inhibition of phospholipase A(2) by dexamethasone, extends the principle to fungi. This study also provides the first evidence of involvement of the lipoxygenase (LOX) pathway rather than the cyclooxygenase (COX) pathway in synthesis of the nodulation mediating eicosanoid(s). The LOX product, 5(S)-hydroperoxyeicosa-6E,8Z,11Z,14Z-tetraenoic acid (5-HPETE), substantially reversed nodulation inhibition caused by dexamethasone and the LOX inhibitors, caffeic acid and esculetin. The COX product, prostaglandin H(2) (PGH(2)), did not reverse the nodulation inhibition by dexamethasone or the COX inhibitor, ibuprofen. None of the inhibitors tested had a significant effect on the phagocytosis of B. bassiana blastospores in vitro. Hemocyte phenoloxidase activity was reduced by dexamethasone, esculetin, and the COX inhibitor, indomethacin. The rescue candidates 5-HPETE and PGH(2) did not reverse the inhibition.     

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 influence insect susceptibility to nucleopolyhedroviruses

Nine pharmaceutical inhibitors of eicosanoid biosynthesis (e.g., bromophenacyl bromide, clotrimazole, diclofenamic acid, esculetin, flufenamic acid, indomethacin, nimesulide, sulindac, tolfenamic acid) that increased the susceptibility of the gypsy moth, Lymantria dispar (L.), to the nucleopolyhedrovirus LdMNPV were tested against the beet armyworm Spodoptera exigua (Hübner), the corn earworm Helicoverpa zea (Boddie) and the fall armyworm Spodoptera frugiperda (J.E. Smith) and their respective NPVs to determine whether these compounds also alter the susceptibility of these insects. The susceptibility of the beet armyworm was increased by six inhibitors (bromophenacyl bromide, clotrimazole, diclofenic acid, esculetin, flufenamic acid, nimesulide). The susceptibility of the fall armyworm was increased by seven inhibitors, (bromophenacyl bromide, diclofenamic acid, esculetin, indomethacin, nimesulide, sulindac, tolfenamic acid), whereas the susceptibility of the corn earworm was increased by only one inhibitor (sulindac). The influence of the cyclooxygenase inhibitor, indomethacin was expressed in a concentration-related manner in beet armyworms. We infer from these findings that eicosanoids, including prostaglandins and lipoxygenase products, act in insect anti-viral defenses.     

Eicosanoids mediate insect hemocyte migration

Hemocyte migration toward infection and wound sites is an essential component of insect defense reactions, although the biochemical signal mechanisms responsible for mediating migration in insect cells are not well understood. Here we report on the outcomes of experiments designed to test the hypotheses that (1) insect hemocytes are able to detect and migrate toward a source of N-formyl-Met-Leu-Phe (fMLP), the major chemotactic peptide from Escherichia coli and (2) that pharmaceutical modulation of eicosanoid biosynthesis inhibits hemocyte migration. We used primary hemocyte cultures prepared from fifth-instar tobacco hornworms, Manduca sexta in Boyden chambers to assess hemocyte migration toward buffer (negative control) and toward buffer amended with fMLP (positive control). Approximately 42% of negative control hemocytes migrated toward buffer and about 64% of positive control hemocytes migrated toward fMLP. Hemocyte migration was inhibited (by >40%) by treating hornworms with pharmaceutical modulators of cycloxygenase (COX), lipoxygenase and phospholipase A2 (PLA2) before preparing primary hemocyte cultures. The influence of the COX inhibitor, indomethacin, and the glucocorticoid, dexamethasone, which leads to inhibition of PLA2, was expressed in a dose-dependent way. The influence of dexamethasone was reversed by injecting arachidonic acid (precursor to eicosanoid biosynthesis) into hornworms before preparing primary hemocyte cultures. The saturated fatty acid, palmitic acid, did not reverse the inhibitor effect. These findings support both our hypotheses, first that insect hemocytes can detect and respond to fMLP, and second, that insect hemocyte migration is mediated by eicosanoids.     

Bacillus pasteurii urease shares with plant ureases the ability to induce aggregation of blood platelets

Ureases (EC 3.5.1.5) are highly homologous enzymes found in plants, bacteria and fungi. Canatoxin, an isoform Canavalia ensiformis urease, has several biological properties unrelated to its ureolytic activity, like platelet-aggregating and pro-inflammatory effects. Here, we describe that Bacillus pasteurii urease (BPU) also induces aggregation of rabbit platelets, similar to the canatoxin-induced effect (ED(50) 0.4 and 0.015 mg/mL, respectively). BPU induced-aggregation was blocked in platelets pretreated with dexamethasone and esculetin, a phospholipase A(2) and a lipoxygenase inhibitor, respectively, while platelets treated with indomethacin, a cyclooxygenase inhibitor, showed increased response to BPU. Methoxyverapamil (Ca(2+) channel blocker) and AMP (ADP antagonist) abrogated urease-induced aggregation, whereas the PAF-acether antagonist Web2170 had no effect. We concluded that platelet aggregation induced by BPU is mediated by lipoxygenase-derived eicosanoids and secretion of ADP from the platelets through a calcium-dependent mechanism. Potential relevance of these findings for bacterium-plant interactions and pathogenesis of bacterial infections are discussed.