46 Lord et al. Archives of Insect Biochemistry and Physiology 51:46–54 (2002) Eicosanoids Mediate Manduca sexta Cellular Response to the Fungal Pathogen Beauveria bassiana: A Role for the Lipoxygenase Pathway Jeffrey C. Lord,1* Sheri Anderson,1 and David W. Stanley2 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 A2 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 dexam- ethasone and the LOX inhibitors, caffeic acid and esculetin. The COX product, prostaglandin H2 (PGH2), 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 PGH2 did not reverse the inhibition. Arch. Insect Biochem. Physiol. 51:46–54, 2002. Published 2002 Wiley-Liss, Inc.† KEYWORDS: eicosanoid; lipoxygenase; Manduca sexta; Beauveria bassiana; nodulation; insect immunity; fungus INTRODUCTION by Stanley-Samuelson, 1994; Stanley and Howard, 1998; Howard and Stanley, 1999; Stanley, 2000). Eicosanoids are oxygenated metabolites of Insect cellular immune responses to microbial arachidonic acid with a broad array of informa- assault are divided into multicellular nodulation tional functions in a diversity of organisms. Among and encapsulation involving both granular cells the roles ascribed to eicosanoids in insects are re- and plasmatocytes, and phagocytosis by individual lease of cricket oviposition behavior (Destephano hemocytes. Several studies have demonstrated a and Brady, 1977; Loher et al., 1981), regulation of role in various insects for eicosanoids in the fluid secretion rates in Malpighian tubules in mos- hemocyte aggregation and nodulation response to quitoes and ants (Petzel and Stanley-Samuelson, blood-borne bacteria (Jurenka et al., 1997; Miller 1992; Van Kerkhove et al., 1995), regulation of et al., 1996; 1999; Tunaz et al., 1999). Mandato et temperature set points in cicadas (Toolson et al., al. (1997) reported that eicosanoids mediate phag- 1994), and modulation of the cellular immune re- ocytosis of carboxylated latex beads, prophenol- sponse to bacteria (Stanley-Samuelson et al., 1991). oxidase activation, and cell spreading in Galleria Eicosanoid actions in cellular defense mechanisms mellonella. With the tobacco hornworm, Manduca have been most intensely investigated (see reviews sexta, and the tenebrionid beetle, Zophobas atratus, 1Grain Marketing and Production Research Center, USDA, ARS, Manhattan, Kansas 2Insect Biochemical Physiology Laboratory, University of Nebraska, Lincoln *Correspondence to: Jeffrey C. Lord, Grain Marketing and Production Research Center, USDA, ARS, 1515 College Avenue, Manhattan, KS 66502. E-mail: [email protected] Received 15 March 2002; Accepted 30 May 2002 Published 2002 Wiley-Liss, Inc. †This article is a US Government work and, as such, is in the public domain in the United StatesArchives of America. of Insect Biochemistry and Physiology DOI: 10.1002/arch.10049 Published online in Wiley InterScience (www.interscience.wiley.com) Role of the Lipoxygenase Pathway 47 as models, Howard et al. (1998) demonstrated that Louis, MO). The lipoxygenase (LOX) inhibitors, the influence of eicosanoids on nodulation applies caffeic acid [3,4-dihydroxycinnamic acid] and es- across bacterial genera. culetin [6,7-dihydroxycoumarin]; the COX inhibi- In this study, we report that the role eicosanoids tor, indomethacin [l-(p-chlorobenzoyl)-5-methoxy- serve in signal transduction for nodulation applies 2-methylindole-3-acetic acid]; the LOX product to the presence of fungi as well as bacteria, as sug- 5-HPETE (5(S)-hydroperoxyeicosa-6E,8Z,11Z,14Z- gested by Dean et al. (2002). We also provide the tetraenoic acid) and the COX product, PGH2 (pros- first evidence that products of the lipoxygenase taglandin H2) were purchased from Biomol (Ply- (LOX) biosynthetic pathway rather than the cyclo- mouth Meeting, PA). Other reagents were obtained oxygenase (COX) pathway are involved in the from Sigma. nodulation events. Nodulation Assay MATERIALS AND METHODS Insects were injected in the third and fourth Organisms prolegs with 26 G Hamilton syringes. Inhibitors were dissolved in ethanol at concentrations of 11– Fifth instar M. sexta larvae weighing 4–6 g were 14 mM. Each injection was in 5 ml volume. The used for all experiments. The larvae were reared at insects were chill-anesthetized and surface disin- 26°C and 75% RH in 15:9 photoperiod from eggs fected with 70% ethanol. Drug treatments were that were purchased from Carolina Biological Sup- injected separately; then fungal blastospores were ply Company (Burlington, NC) or North Carolina injected within 2 min. In some experiments, the State University. They were cultured on an artifi- influence of the pharmaceuticals was potentially cial diet modified from Baumhover (1985). The rescued by injecting candidate rescue compounds diet contained sorbic acid, methyl-p-hydroxy- within 2 min of the fungal challenge. After injec- benzoate, and streptomycin sulfate as preservatives. tions, the insects were incubated for 2 h at room Beauveria bassiana isolate GHA was obtained temperature. They were then bled through a from Mycotech Corp. (Butte, MT). It was main- clipped proleg into iced test tubes, and 250 ml of tained in Sabouraud dextrose broth throughout the hemolymph were combined with 100 ml of anti- experiments by repeated mycelium-blastospore coagulant saline solution containing 98 mM transfers (Rombach et al., 1988). All experiments NaOH, 146 mM NaCl, 17 mM EDTA, and 41 mM were conducted with blastospores that were sepa- citric acid (Mead et al., 1986). The hemolymph rated from mycelium by passage through glass was placed in CoverWell imaging chambers (Grace wool, washed twice by low speed centrifugation Bio-Labs, Bend, OR) and all nodules in 250 ml in distilled, deionized water, and adjusted to 4 ´ were counted with an inverted microscope. The 7 10 /ml for injections. hemolymph of five insects per treatment was handled separately in four trials for all treatments Reagents except for three trials with esculetin and escule- tin with 5-HPETE. The phospholipase A2 (PLA2) inhibitor, dex- amethasone [(11b,16a)-9-fluoro-11,17, 21-trihy- Phenoloxidase Assay droxy-16-methylpregna-1,4-diene-3,20-dione]; the cyclooxygenase (COX) inhibitor, ibuprofen Hemolymph from 12 chilled fifth instar larvae [a-methyl-4-(isobutyl)phenylacetic acid]; L-DOPA was collected into individual test tubes containing (L-3,4-dihydroxyphenylalanine); and arachidonic anticoagulant saline. Hemocytes were washed in acid [5,8,11,14-eicosatetraenoic acid] were pur- anticoagulant saline twice by centrifugation at 100g chased from Sigma Chemical Company (St. for 5 min and resuspended in Manduca buffered September 2002 48 Lord et al. saline (MBS) (Willot et al., 1994). Ten microliters were removed from the slides, and coverslips were of 10 mM test compound in ethanol were added added and sealed. Internalization of blastospores to 1 ml of hemocyte suspension and incubated at was scored by fluorescence microscopy on 100 room temperature for 1 h. The samples were then hemocytes per well. Each treatment was applied placed in ice and sonicated for 15 sec at 30% to three wells, and the experiment was carried out power with a VirSonic 475 probe sonicator (Virtis, three times. Gardiner, NY). Cellular debris was removed by cen- trifugation at 13,300g for 20 min at 4°C. The su- Statistical Analyses pernatant protein was determined by bicinchonic acid assay (Pierce, Rockford, IL). The hemocyte Significant treatment effects were identified by lysate supernatants were distributed in 100-ml one-way analysis of variance at a = 0.05 using 7 aliquots in a 96-well plate with 50 ml of 10 B. GraphPad InStat (GraphPad Software, San Diego). bassiana blastospores/ml and incubated for 30 min The nodulation data of individual assays were not at 37°C. Fifty microliters of 3 mg/ml L-DOPA was significantly different and were pooled for analy- added to each well and the absorbance was read sis. The phagocytosis and phenoloxidase (PO) data at 490 nm for 60 min. Units of PO activity were varied significantly among assays, and arcsine trans- calculated as 0.001 D absorbance/mg protein/min formed trial means were used in the analysis. Dif- and all results were analyzed as proportions of un- ferences among means were determined by the treated controls. The experiment was carried out Student-Newman-Keuls procedure. three times. RESULTS Phagocytosis Assay Nodulation Hemocytes from 12 larvae were collected and washed as in the PO assay. The suspensions were Dexamethasone significantly reduced the nodu- adjusted to 106 hemocytes/ml and dispensed in 170 lation response to B. bassiana, from about 450 nod- ml aliquots into the wells of chamber slides (Nalge ules/ml hemolymph in control to about 200 Nunc, Naperville, IL). Test compounds were dis- nodules/ml hemolymph in dexamethasone-treated