A secretory PLA2 associated with tobacco hornworm hemocyte membrane preparations acts in cellular immune reactions

We report on a secretory phospholipase A2 (sPLA2) associated with membrane-enriched fractions prepared from hemocytes of the tobacco hornworms, Manduca sexta. Virtually no PLA2 activity was detected in serum of immunologically naive or bacterially challenged hornworms. PLA2 activity was detected in cytosolic and membrane-enriched fractions prepared from hemocytes. PLA2 activity in the cytosolic fraction (1.2 pmol/mg/h) was approximately 4-fold greater than in the membrane-enriched fraction. The cytosol-associated PLA2 activity was strongly inhibited in reactions conducted in the presence of the specific cytosolic PLA2 inhibitor methylarachidonyl fluorophosphate (MAFP) but not in the presence of the sPLA2 inhibitor p-bromophenacyl bromide (BPB). Conversely, the membrane-associated PLA2 activity was inhibited in reactions conducted in the presence of BPB but not in the presence of MAFP. While the cytosol-associated PLA2 was independent of calcium, the membrane-associated sPLA2 required calcium for full catalytic activity. Hornworms treated with either BPB, MAFP or the glucocorticosteroid dexamethasone were severely impaired (by 50 to 80% relative to controls) in their ability to form nodules in reaction to bacterial challenge. However, the immune-impairing influence of the inhibitors was reversed by treating larvae with arachidonic acid, a precursor for eicosanoid biosynthesis. We infer that the biological significance of the sPLA2 (as well as the previously characterized cytosolic PLA2) relates to hydrolysis of polyunsaturated fatty acids from cellular phospholipids. Moreover, this enzyme may be the target of immunity-impairing factors from the bacterium Xenorhabdus nematophila. The fatty acids serve as precursors for the generation of eicosanoids responsible for mediating and coordinating cellular immune reactions to infection.     

Lipopolysaccharide evokes microaggregation reactions in hemocytes isolated from tobacco hornworms, Manduca sexta

Insect cellular immune reactions to bacterial infection include nodule formation. Eicosanoids mediate several cellular actions in the nodulation process, including formation of hemocyte microaggregates, an early step. In previous work, we reported that isolated hemocytes produce and secrete eicosanoids that influence hemocyte behavior in response to bacterial challenge. We also reported that microaggregate formation in response to challenge was mediated by prostaglandins (PGs), but not by products of the lipoxygenase (LOX) pathways. In this paper we describe experiments designed to test the idea that exposing isolated hemocytes to lipopolysaccharide (LPS) evokes formation of hemocyte microaggregates and this cellular action is mediated by PGs. Results show that isolated hemocyte preparations challenged with LPS formed more hemocyte microaggregates than unchallenged preparations (6.9x10(3) microaggregates/ml hemolymph vs. 2.5x10(3) microaggregates/ml hemolymph). LPS challenge stimulated formation of hemocyte microaggregates in a dose dependent manner. Experimental groups pretreated with cyclooxygenase inhibitors produced fewer hemocyte microaggregates in response to LPS challenge than untreated control groups. The formation of hemocyte microaggregates was not influenced by LOX inhibitors. Furthermore, the influence of dexamethasone was reversed by supplementing the experimental groups with the eicosanoid precursor fatty acid molecule, arachidonic acid and PGH(2). Palmitic acid, which is not substrate for eicosanoid biosynthesis, did not reverse the effects of dexamethasone on the formation of microaggregates. The LOX product 5(S)hydroperoxyeicosa-6E,8Z,11Z,14Z-tetraenoic acid also did not reverse the effects of dexamethasone. These results are consistent with similar investigations performed with bacterial suspensions. We infer that isolated hemocyte preparations recognize and react to LPS by forming microaggregates and this reaction is mediated by PGs, but not products of the LOX pathway.     

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.     

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.     

An entomopathogenic bacterium, Xenorhabdus nematophila, inhibits hemocytic phospholipase A2 (PLA2) in tobacco hornworms Manduca sexta

The entomopathogenic bacterium, Xenorhabdus nematophila, induces immunodepression in target insects and finally leads to lethal septicemia of the infected hosts. A hypothesis has been raised that the bacteria inhibit eicosanoid-biosynthesis pathway to interrupt immune signaling of the infected hosts. Here, we show direct evidence that X. nematophila inhibits the activity of phospholipase A2 (PLA2), the initial step in the eicosanoid-biosynthesis pathway. Inhibition of PLA2 was dependent on both incubation time with X. nematophila and the bacterial concentration in in vitro PLA2 preparations of Manduca sexta hemocytes. While living bacteria inhibited PLA2 activity, heat-killed X. nematophila rather increased PLA2 activity. X. nematophila secreted PLA2 inhibitor(s) which were detected in the organic, but not aqueous, extract of the bacterial culture medium. The PLA2 inhibitory activity of the organic extract was lost after heat treatment. These results clearly indicate that X. nematophila inhibits PLA2 activity, and thereby inhibits eicosanoid biosynthesis which leads to immunodepression of the infected hosts.     

Eicosanoid biosynthesis is activated via Toll, but not Imd signal pathway in response to fungal infection

Phospholipase A(2) (PLA(2)) catalyzes hydrolysis of phospholipids at sn-2 position and usually releases arachidonic acid, which is oxygenated into various eicosanoids that mediate innate immune responses in insects. PLA(2) activities were measured in both immune-associated tissues of hemocyte and fat body in the beet armyworm, Spodoptera exigua. Upon challenge of an entomopathogenic fungus, Beauveria bassiana, the PLA(2)s were significantly activated in both hemocyte and fat body. The fungal infection also induced gene expression of antimicrobial peptides (AMPs), such as two attacins, cecropin, gallerimycin, gloverin, hemolin, and transferrin of S. exigua. RNA interference of Toll or Imd signal pathway using double-stranded RNAs (dsRNAs) specific to SeToll or SeRelish suppressed specific AMP gene expressions, in which dsRNA specific to SeToll suppressed two attacins, cecropin, gallerimycin, gloverin, hemolin, and transferrin I, while dsRNA specific to SeRelish suppressed only cecropin. Interestingly, dsRNA specific to SeToll also significantly inhibited the activation of PLA(2) in response to the fungal infection, but dsRNA specific to SeRelish did not. Eicosanoid-dependent hemocyte nodulation was inhibited by dsRNA specific to SeToll but was not by dsRNA specific to SeRelish. These results suggest that eicosanoid biosynthesis is activated via Toll, but not Imd signal pathway in response to fungal infection in S. exigua.     

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.     

Up-regulation of circulating hemocyte population in response to bacterial challenge is mediated by octopamine and 5-hydroxytryptamine via Rac1 signal in Spodoptera exigua

Bacterial challenge induced a significant increase in the total hemocyte population within 4 h in the beet armyworm, Spodoptera exigua. Octopamine and 5-hydroxytryptamine (5-HT) are known to play critical roles in mediating insect immune responses. This study analyzed the effects of both biogenic monoamines on mediating up-regulation of circulating hemocyte population in response to bacterial challenge. Injection of either octopamine or 5-HT induced a significant increase in the total hemocyte count in the hemolymph without any bacterial challenge. On the other hand, the monoamine antagonists, phentolamine (an octopamine antagonist) and ketanserin (a 5-HT antagonist) each suppressed the increase of the circulating hemocyte counts in response to bacterial challenge. This rapid change of circulating hemocyte population did not appear to be the result of de novo hemocyte production from the hematopoietic organ because a physical block (“ligation”) of hemolymph circulation between thorax and abdomen did not inhibit the increase of hemocyte counts in the isolated abdomen in response to bacterial challenge. The effects of the two monoamines on hemocyte numbers were not dependent on the mediatory effects of eicosanoids, because dexamethasone, an eicosanoid biosynthesis inhibitor, had no effect on the hemocyte recruitment induced by the monoamines. On the other hand, an adenylate cyclase inhibitor, NKY80, significantly impaired hemocyte mobilization in response to bacterial challenge, implying involvement of cyclic AMP in the control of hemocyte numbers. Also, a Rac1 inhibitor, NSC23766, significantly antagonized the effects of monoamines in increasing circulating hemocyte numbers. Rac1 activity was necessary to form F-actins in the hemocytes of S. exigua, where its activity showed a quantitative correlation with hemocyte-spreading behavior. This study suggests that octopamine and 5-HT mediate a rapid increase of circulating hemocyte population in response to bacterial challenge via Rac1 signal in S. exigua.     

Lipid mediators and vector infection: Trypanosoma rangeli inhibits Rhodnius prolixus hemocyte phagocytosis by modulation of phospholipase A2 and PAF-acetylhydrolase activities

In this work we investigated the effects of Trypanosoma rangeli infection through a blood meal on the hemocyte phagocytosis in experiments using the 5th instar larvae of Rhodnius prolixus. Hemocyte phagocytic activity was strongly blocked by oral infection with the parasites. In contrast, hemocyte phagocytosis inhibition caused by T. rangeli infection was rescued by exogenous arachidonic acid (20 microg/insect) or platelet activating factor (PAF; 1 microg/insect) applied by hemocelic injection. Following the oral infection with the protozoan we observed significant attenuation of phospholipase A2 (PLA2) activities in R. prolixus hemocytes (cytosolic PLA2: cPLA2, secreted PLA2: sPLA2 and Ca+2-independent PLA2: iPLA2) and enhancement of sPLA2 activities in cell-free hemolymph. At the same time, the PAF-acetyl hydrolase (PAF-AH) activity in the cell-free hemolymph increased considerably. Our results suggest that T. rangeli infection depresses eicosanoid and insect PAF analogous (iPAF) pathways giving support to the role of PLA2 in the regulation of arachidonic acid and iPAF biosynthesis and of PAF-AH by reducing the concentration of iPAF in R. prolixus. This illustrates the ability of T. rangeli to modulate the immune responses of R. prolixus to favor its own multiplication in the hemolymph.     

Genes encoding phospholipases A2 mediate insect nodulation reactions to bacterial challenge

We propose that expression of four genes encoding secretory phospholipases A₂ (sPLA₂) 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 PLA₂-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 sPLA₂ 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 sPLA₂ and expressed them in E. coli cells to demonstrate these genes encode catalytically active sPLA₂s. The recombinant sPLA₂s were inhibited by sPLA₂ inhibitors. Injecting larvae with double-stranded RNAs specific to each of the five genes led to reduced expression of the corresponding sPLA₂ 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 sPLA₂s mediates nodulation reactions. A polyclonal antibody that reacted with all five sPLA₂s showed the presence of the sPLA₂ enzymes in hemocytes and revealed that the enzymes were more closely associated with hemocyte plasma membranes following infection. Identifying specific sPLA₂ genes that mediate nodulation reactions strongly supports our hypothesis that sPLA₂s are central enzymes in insect cellular immune reactions.     

Phospholipase A2 in hemocytes of the tobacco hornworm,Manduca sexta

On the hypothesis that prostaglandins and other eicosanoids mediate nodulation responses to bacterial infections in insects, we describe an intracellular phospholipase A₂ (PLA₂) in homogenates prepared from hemocytes collected from the tobacco hornworm, Manduca sexta. PLA₂ hydrolyzes fatty acids from the sn-2 position of phospholipids. Some PLA₂s are thought to be the first and rate-limiting step in biosynthesis of prostaglandins and other eicosanoids. The hemocyte PLA₂ activity was sensitive to hemocyte homogenate protein concentration (up to 250 μg protein/reaction), pH (optimal activity at pH 8.0), and the presence of a Ca²⁺ chelator. Like PLA₂s from mammalian sources, the hemocyte PLA₂ was inhibited by the phospholipid analog oleyoxyethyl phosphorylcholine. Whereas most intracellular PLA₂s require Ca²⁺ for catalytic activity, some PLA₂s, including the hemocyte enzyme, are Ca²⁺-independent. The hemocyte PLA₂ exhibited a preference for arachidonyl-associated substrate over palmitoyl-associated substrate. These findings show that M. sexta hemocytes express a PLA₂ that shows a marked preference for hydrolyzing arachidonic acid from phospholipids. The biological significance of this enzyme relates to cellular immune responses to bacterial infections. The hemocyte PLA₂ may be the first biochemical step in synthesis of the eicosanoids that mediate cellular immunity in insects. © 1996 Wiley-Liss, Inc.     

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.     

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 20.0 kDa, 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 48 h 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, PGE₂. 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.     

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.     

Two groups of entomopathogenic bacteria, Photorhabdus and Xenorhabdus, share an inhibitory action against phospholipase A2 to induce host immunodepression

Photorhabdus and Xenorhabdus are two genera of entomopathogenic bacteria having a mutualistic relationship with their respective nematode hosts, Heterorhabditis and Steinernema. One of the pathogenic mechanisms of these bacteria includes host immunodepression, which leads to lethal septicemia. It has been known that X. nematophila inhibits phospholipase A2 (PLA2) to induce host immunodepression. Here, we tested the hypothesis of PLA2 inhibition using another bacterial species involved in other genera. P. temperata subsp. temperata is the intestinal symbiont of an entomopathogenic nematode, H. megidis. The bacteria caused potent pathogenicity in a dose-dependent manner against the fifth instar larvae of a test target insect, Spodoptera exigua, as early as 24 h after the intra-hemocoelic injection. In response to the live bacterial injection, hemocyte nodulation (a cellular immune response) and prophenoloxidase (pPO) activation were inhibited, while the injection of heat-killed bacteria significantly induced both immune reactions. The immunodepression induced by the live bacteria was reversed by the addition of arachidonic acid, the catalytic product of phospholipase A2. In contrast, the addition of dexamethasone, a specific PLA2 inhibitor to the heat-killed bacterial treatment, inhibited both immune capacities. In addition to a previously known PLA2 inhibitory action of X. nematophila, the inhibition of P. temperata temperata on PLA2 suggests that bacteria symbiotic to entomopathogenic nematodes share a common pathogenic target to result in an immunodepressive state of the infected insects. To prove this generalized hypothesis, we used other bacterial species (X. bovienni, X. poinarii, and P. luminescens) involved in these two genera. All our experiments clearly showed that these other bacteria also share their inhibitory action against PLA2 to induce host immunodepression.     

Kinetics of phospholipase A2, arachidonic acid, and eicosanoid appearance in mouse zymosan peritonitis

Intraperitoneal injection of zymosan into mice induces a peritonitis characterized by cellular influx, plasma leakage and the appearance of arachidonic acid (AA) metabolites. We report that zymosan injection also stimulates the accumulation of AA, docosahexaenoic acid, linoleic acid, and phospholipase A2 (PLA2) activity. The amount of the unsaturated fatty acids (UnFA) varies both with the zymosan dose and time. Significantly increased levels of UnFA were first detected 15 min after zymosan injection. Maximal levels of the UnFA were reached 1 to 2 h post zymosan injection (AA: 725 +/- 29 ng/mouse, docosahexaenoic acid: 296 +/- 23 ng/mouse, linoleic acid: 4489 +/- 179 ng/mouse) and declined to saline control levels by 8 h. PLA2 activity was significantly increased 5 to 15 min after zymosan injection. Maximal levels of PLA2 activity occurred 15 to 30 min after zymosan injection (31.8 +/- 9.1 nmol phospholipid/mg protein/h) and then decreased by 30% through 24 h. Neither the appearance of UnFA nor PLA2 activity correlated with cellular influx, but both were coincident with plasma exudation at 5 to 15 min after zymosan. However, maximal exudation occurred 1 to 2 h post zymosan injection similar to that seen with the UnFA but not PLA2. These latter results suggest that a significant portion of the UnFA found in the peritoneal cavity of zymosan-injected mice originates from the plasma. PLA2 activity at the early time points (5 to 15 min) may also contribute to the levels of UnFA via hydrolysis of tissue and/or cellular phospholipids.     

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.     

Benzylideneacetone, an immunosuppressant, enhances virulence of Bacillus thuringiensis against beet armyworm (Lepidoptera: Noctuidae)

Benzylideneacetone (BZA) is a metabolite of gram-negative entomopathogenic bacterium Xenorhabdus nematophila, and it acts as an enzyme inhibitor against phospholipase A2 (PLA2). PLA2 catalyzes a committed biosynthetic step of eicosanoids, which mediate insect immune reactions to infection by microbial pathogens. This study tested a hypothesis that a putative immunosuppressive activity of BZA may enhance virulence of Bacillus thuringiensis against the fifth instars of Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae). In in vitro conditions, BZA significantly inhibited hemocyte microaggregation induced by B. thuringiensis and impaired hemocyte-spreading behavior of S. exigua in a dose-dependent manner. Oral administration of BZA gave similar immunosuppressive effect on the hemocytes of the fifth instars. Although BZA itself did not possess any insecticidal activity on oral administration, when BZA was treated in a mixture with a low dose of B. thuringiensis spp. aizawai to fifth instars, the bacterial virulence was significantly enhanced. BZA also enhanced virulence of B. thuringiensis spp. kurstaki, which alone was of limited effectiveness against S. exigua. This study suggests that an immunosuppression by BZA is positively linked to potentiation of B. thuringiensis.