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Eicosanoids mediate nodulation reactions to bacterial infections in adults of two 17-year periodical cicadas, Magicicada septendecim and M. cassini
Journal of Insect Physiology 45 (1999) 923–931 www.elsevier.com/locate/ibmbjip
Eicosanoids mediate nodulation reactions to bacterial infections in adults of two 17-year periodical cicadas, Magicicada septendecim and M. cassini Hasan Tunaz, Jon C. Bedick, Jon S. Miller, W. Wyatt Hoback, Rico L. Rana, David W. Stanley * Insect Biochemical Physiology Laboratory, University of Nebraska, Lincoln, NE 68583-0816, USA Received 17 August 1998; accepted 11 December 1998
Abstract 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 A2, 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. 1999 Elsevier Science Ltd. All rights reserved. Keywords: Cellular immunity; Eicosanoid biosynthesis; Hemiptera; Phospholipase A2
1. Introduction We have been investigating the idea that eicosanoids mediate cellular defense reactions, particularly nodulation, to bacterial infections in insects. Eicosanoid is a collective term for all oxygenated metabolites of three C20 polyunsaturated fatty acids, specifically 20:3n-6, 20:4n-6 and 20:5n-3. Three main groups of eicosanoids are recognized (Fig. 1; Stanley-Samuelson, 1994a). Prostaglandins and thromboxanes are products of the cyclooxygenase pathways. Epoxyeicosatrienoic acids are products of cytochrome P450s, often called epoxygenase pathways for convenience. The many lipoxygenase systems yield a complex assemblage of products, including
* Corresponding author. Tel.: +1-402-472-8710; fax: +1-402-4728746. E-mail address: DStanley얀unlnotes.unl.edu (D.W. Stanley)
leukotrienes. Various eicosanoids are present and biologically active in virtually all mammalian tissues and body fluids. Eicosanoids also exert many actions in invertebrates, including aspects of reproduction, ion transport, and host–parasite relationships, all detailed in recent reviews (Stanley-Samuelson, 1994a,b; Stanley and Howard, 1998; Stanley, 1999). Nodulation is a cellular reaction which clears large numbers of bacterial cells from circulation during the early phase of an infection in tobacco hornworms, typically the first 2 hours (Horohov and Dunn, 1983). Nodulation is a complex process beginning with microaggregations of hemocytes (Gupta, 1991; Beckage et al., 1993; Wiesner et al., 1998). Bacterial cells are entrapped within the microaggregates. The microaggregates continue to grow by attracting hemocytes (presumably by chemotaxis, as seen in mammalian systems) and attached bacteria. The process ends with a phase of melanization, which leaves darkened nodules, typically about 0.1 mm
0022-1910/99/$ - see front matter. 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 2 - 1 9 1 0 ( 9 9 ) 0 0 0 7 2 - 4
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Fig. 1. An outline of eicosanoid biosynthesis as understood from research on mammalian systems. Three C20 polyunsaturated fatty acids, 20:3n6, 20:4n-6 and 20:5n-3 are potential substrates for eicosanoid biosynthesis, of which metabolism of arachidonic acid is best understood. Chemical structures are denoted by numerals and major biosynthetic pathways are indicated by capital letters. 1=a cellular phospholipid. 2=hydrolyzed arachidonic acid. 3=prostaglandin E2. 4=5-hydroperoxyeicosatetraenoic acid. 5=leukotriene B4. 6=11,12-epoxyeicosatrienoic acid. 7=lipoxin A. A=phospholipase A2; B=cyclooxygenase and associated enzyme steps; C=cytochrome P450 epoxygenase; D=lipoxygenase.
in diameter, attached to various organs and body walls. Hence, bacterial cells are cleared from circulation in a topological sense by mass entrapments. We tested the idea that eicosanoids mediate nodulation reactions to bacterial infections in tobacco hornworms (Miller et al., 1994). Outcomes of these experiments showed that inhibition of eicosanoid biosynthesis immediately prior to bacterial infections severely impaired the ability of the hornworms to form microaggregates and nodules in response to bacterial infections.
Using similar experimental protocols, we have shown that microaggregation and nodulation reactions to bacterial infections depend on eicosanoid biosynthesis in the tenebrionid beetle, Zophobas atratus (Miller et al., 1996), in the silkworm, Bombyx mori (Stanley-Samuelson et al., 1997), and in two other moths, black cutworms Agrotis ipsilon and true armyworms Pseudaletia unipuncta (Jurenka et al., 1997). Mandato et al. (1997) showed that cell spreading, a distinct phase of nodulation, prophenyloxidase activation, and phagocytosis are mediated by eicosanoids in waxmoths, Galleria mel-
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lonella. All experiments have so far produced results supporting the idea that eicosanoids mediate nodulation reactions to bacterial challenges in insects, which we now formalize as the eicosanoid hypothesis. Most recently we tested the hypothesis in an exopterygote insect, adults of the cricket Gryllus assimilis (Miller et al., 1999). Taken together, these findings strongly support the eicosanoid hypothesis. Nevertheless, as a single species taken from a rather large assemblage of insects, it is difficult to extrapolate from the cricket G. assimilis to the remaining orders within the Division Exopterygota. The extrapolation is more speculative because the orders within this Division are phylogenetically diverse (Daly et al., 1998). The cricket is an orthopteran grouped within the Superorder Orthopteroidea. The other main Superorder is the Hemipteroidea, a large monophyletic group completely distinct from the Orthopteroidea (Daly et al., 1998). We recognize that the eicosanoid hypothesis is only weakly supported by studies of a single orthopteroid insect species. Hence, we deemed it necessary to test the eicosanoid hypothesis in a representative of the Hemipteroidea. The biology of hemipteroids differs in important ways from all other insects we have tested so far. Hemipteriod species are fluid-feeding insects during all post-embryonic life stages, and they exhibit many unusual life history patterns. One group of hemipteroids, the periodical cicadas are remarkably specialized insects which spend extended larval periods underground and short adult phases above ground. Another good reason to select cicadas as an experimental insect is that eicosanoids have been implicated in another area of cicada physiology. Toolson et al. (1994) suggested that eicosanoids regulate the thermoregulatory set points in the desert cicada, Tibicen dealbatus. If this is so for other cicadas, including periodical cicadas, we can point to the possibility of two distinct roles of eicosanoids in cicadas: one in thermobiology and the second in immunity. Multiple eicosanoid actions are commonly recognized in mammals, and have been postulated for silkmoths, B. mori. Prostaglandins release egg-laying behavior, modulate release of lipids from fat body and mediate nodulation reactions to infections in this species (Stanley-Samuelson et al., 1997). As seen in mammals, invertebrates probably rely on eicosanoid actions that operate independently in many different physiological systems within the same organism. Hence, adult periodical cicadas present an opportunity to test the hypothesis that eicosanoids mediate cellular immune reactions in the Hemipteriodea. In this paper, we report that eicosanoids mediate nodulation reactions to bacterial infections in adults of two hemipteran insects, the 17-year periodical cicadas, Magicicada septendecim and M. cassini. We show that adults of these species form nodules in response to bacterial infections, and that the nodulation response is sev-
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erely impaired by treating the cicadas with eicosanoid biosynthesis inhibitors prior to infection.
2. Materials and methods 2.1. Organisms We collected adult cicadas, M. septendecim and M. cassini within one week of adult emergence from Schramm State Park, in eastern Nebraska. After identifying males and females to species on morphological grounds (Moore and Alexander, 1958), we held them overnight in a laboratory refrigerator. The insects were removed to room temperature and allowed to recover normal activity before experimental manipulations. Cultures of a nonpigmented strain of Serratia marcescens and nutrient broth (Difco) were purchased from Carolina Biological Supply (Burlington, NC). Bacteria were grown in 50 ml of nutrient broth in an environmental shaker at 37°C and 100 rpm. Bacteria were grown to a titre of 107 colony forming units (cfu)/ml, and used in stationary phase. 2.2. Injections and assays for nodulation Test insects were injected with either the PLA2 inhibitor dexamethasone [(11β, 16α)-9-fluoro-11,17,21-trihydroxy-16-methylpregna-1,4-dione], one of the cyclooxygenase inhibitors indomethacin [1-p-chlorobenzoyl)-5methoxy-2-methyl-3-indolyl-acetic acid], naproxin [D-2(6-methoxy-2-naphthyl)propionic acid], or the 5- and 12lipoxygenase inhibitor esculetin [6,7-dihydroxycoumarin] (all inhibitors from BioMol, Plymouth Meeting, PA). In some experiments, cicadas were also injected with arachidonic acid [5,8,11,14-eicosatetraenoic acid] (Sigma Chemical Co., St. Louis, MO). Control cicadas were injected with nutrient broth, or 95% ethanol. All injections of pharmaceuticals were in a standard dosage of 26 µg in 10 µl of ethanol, except in dose–response experiments. The fatty acids were injected at dosages of 50 µg in 5 µl of ethanol per adult cicada. Prior to injections, cicadas were surface sterilized by swabbing their surfaces with 95% ethanol. Insects were infected by injecting a bacterial dosage of 106 cfu/cicada (Miller et al., 1994, 1996) in 100 µl aliquots using a 26 gauge 0.5⬙ needle attached to a 1000 µl syringe. Drugs and control substances were injected into the opposite lateral side of the abdomen in 10 µl volumes using a 10 µl Hamilton 701 syringe. Nodulation was assessed at selected times post-infection (PI). Cicadas were anesthetized by chilling on ice, then the hemocoels were exposed. Melanized, dark nodules were counted under a stereomicroscope at 60×. The nodules were distinct, and counting reliably reflected the extent on the nodulation response to infections (Miller
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et al., 1994, 1996). The gonads, fat bodies and other internal tissues were carefully probed for previously unseen nodules. We considered the possibility that nodulation reactions to bacterial infections in M. septendecim and M. cassini might be quantitatively different. After routine bacterial challenges, both species formed similar numbers of nodules, 35±4.4/individual for M. septendecim males and 38±3 nodules/individual for M. cassini males (n=5 for each species, not significantly different by Student’s t test). We used males of the larger M. septendecim for most manipulations, but approximately 10% of the individuals were males of M. cassini. The data for both species were combined for presentation, and the implications of the experimental results apply to both species. 2.3. Time-course of nodulation: influence of dexamethasone Individuals in two groups of male cicadas were injected with 10 µl of ethanol or with 26 µg of dexamethasone in 10 µl of ethanol. The cicadas were immediately injected with bacteria as described. At 2, 4, 8, and 16 h PI, sub-groups of control and experimental insects were anesthetized and nodulation assessed. 2.4. Dose–response curve for dexamethasone We conducted two separate experiments, one with males and one with females. Individuals in four groups of cicadas were injected with 10 µl of ethanol, or with 0.26, 2.6 or 26 µg of dexamethasone in 10 µl of ethanol, then injected with a standard dosage of bacteria. At 8 h PI, the cicadas were anesthetized, and nodulation was assessed. Males and females differ in internal anatomy. Most of the abdomen of males is as a hollow resonance chamber, while the abdomens of females are filled with fat body and well-developed ovaries. Because of these anatomical differences we used male cicadas in all remaining experiments because the differences facilitated assessment of nodulation. 2.5. Fatty acid rescue experiments Individuals in two groups of cicadas were injected with either 10 µl of ethanol or 26 µg of dexamethasone in 10 µl of ethanol, then injected with bacteria as described. Immediately after infection, the dexamethasone-treated cicadas were divided into two subgroups. Individuals in one sub-group were treated with 50 µg of arachidonic acid in 5 µl of ethanol. Individuals in another sub-group were treated with 5 µl of ethanol to control for the effects of the extra injection on nodulation. At 8 h PI, the cicadas were anesthetized and nodulation was assessed.
2.6. Influence of other eicosanoid biosynthesis inhibitors on nodulation Cicadas were divided into groups and injected with either one of the cyclooxygenase inhibitors indomethacin or naproxin, or the lipoxygenase inhibitor esculetin, all in 10 µl of ethanol. Control insects were injected with 10 µl of ethanol. Following injections, the cicadas were infected with a standard dosage of bacteria as described. At 8 h PI, the cicadas were anesthetized and nodulation was assessed. 2.7. Eicosanoid biosynthesis by cicada fat body preparations We investigated prostaglandin (PG) biosynthesis by microsomal-enriched preparations of fat body from adult cicadas. These experiments followed protocols developed for fat body from M. sexta (Stanley-Samuelson and Ogg, 1994). Briefly, fat body was removed from chilled adult male and female cicadas, then homogenized in a glass homogenizer. The homogenates were sonicated for 10 s at 40 W using a VibraCell sonicator (VibraCell, Danbury, CT). This preparation was centrifuged for 10 min at 735g, and the supernatant was centrifuged for another 20 min at 16,000g, both steps at 4°C. The 16,000g microsomal-enriched supernatants were used in all experiments. Protein concentrations in these preparations were determined against bovine serum albumin using the bicinchoninic acid reagent (Pierce, Rockford, IL). Standard curves and samples were read on a BioTek microtitre plate reader at 562 nm. Radioactive arachidonic acid (5,6,8,9,11,12,14,15-3H20:4, 60–100 Ci/mmol) was purchased from DuPont. The incubation buffer was 0.05 M KH2PO4, pH 8.0, amended with a standard co-factor mixture (2.4 mM reduced glutathione, 0.25 mM hydroquinone and 25 µg hemoglobin). For each PG biosynthesis reaction, 0.4 µCi of labeled arachidonic acid was dispensed into reaction tubes and the solvent was evaporated. The reactions were carried out in 1.0 ml total volume. The experiments were preceded by a 10 min pre-incubation with all reaction components, except the protein source. The protein source was added (1.5 mg/ml in each reaction), and after a 2 min reaction period, the reactions were stopped by addition of 500 µl 0.1 N HCl. Reaction products were extracted from the acidified reaction mixture three times in ethyl acetate. The combined extracts, containing PGs and possibly lipoxygenase products, were evaporated under N2. A mixture of appropriate eicosanoid standards was added to each sample, then samples were applied to thin-layer chromatography plates. The plates were developed and fractions observed as described previously (Stanley-Samuelson and Ogg, 1994). Bands corresponding in Rf to selected authentic eicosanoid standards and to free fatty acids were transferred to liquid
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scintillation vials. Radioactivity in each fraction was determined by adding Ecolite scintillation mixture (ICN Biomedicals, Irvine, CA) and counting on a LKB Wallac 1209 Rackbeta Liquid Scintillation Counter (Pharmacia, Turku, Finland) at 50% counting efficiency for 3H. Eicosanoid biosynthesis was calculated from the liquid scintillation data. In negative control experiments, microsomal-enriched preparations were heated in boiling water for 15 min before the experiments, and processed as just described. The results of these control experiments were used to correct values from biosynthesis experiments as described (Stanley-Samuelson and Ogg, 1994). 2.8. Assessing digestive phospholipase A2 in midgut contents We used standard procedures to assess digestive PLA2 activity (Rana et al., 1998; Nor Aliza and Stanley, 1998). Digestive tracts were dissected from males and females of M. septidecim, homogenized in buffer (0.1 M Tris, 5 mM CaCl2, pH 9.0), then centrifuged at 11,750g for 10 min. The resulting supernatants were used as buffer-soluble enzyme preparations, which were held on ice until further use. Protein concentrations in the supernatants were determined as described in the preceding section. Radioactive phosphatidylcholine (1-palmitoyl, 2-arachidonyl [arachdonyl-1-14C]; 1.9 GBq/mmol; New England Nuclear, Wilmington, DE) was prepared in vesicles by vigorously vortexing in buffer. The final radioactive substrate concentration was 0.25 µCi/250 µl reaction volume. The PLA2 reactions were initiated by adding aliquots of enzyme source (2.5 mg protein) to tubes containing substrate vesicles. After 30 min incubations at 28°C, the reactions were terminated by adding 0.5 ml of chloroform:methanol, 2:1, v/v, acidified with 50 µl 2 M HCl. Cold arachidonic acid was added to serve as a carrier. Two additional extraction steps using 0.5 ml chloroform followed. Free fatty acids were isolated by TLC plates developed in petroleum ether:ethyl ether:acetic acid (80:20:1, v/v). Fractions corresponding to free fatty acids and to phospholipids were transferred to liquid scintillation vials. Radioactivity in each vial was determined as described above. 2.9. Statistical analysis Data on nodulation were analyzed by ANOVA and significance was determined at P⬍0.05 using the Least Significant Difference (LSD) test. 3. Results 3.1. Time course of nodulation The time course of visible nodule formation in two groups of cicadas is displayed in Fig. 2. The ethanol-
Fig. 2. Time course of nodulation in adult male cicadas in response to intrahemocoelic infections with the pathogenic bacterium S. marcescens. Test insects were first injected with dexamethasone (unfilled circles), and control insects were first injected with ethanol (filled circles). Within 3–10 min, both groups of insects were then intrahemocoelically infected with approximately 1×106 bacterial cells. At the indicated times PI, the insects were anesthetized on ice and nodulation assessed. Each point indicates the mean number of nodules found in each insect, and the error bars represent 1 SEM (n=5 individuals for each point).
treated control insects produced about 20 nodules/cicada at 2 h PI, which increased to nearly 60 nodules/cicada by 8 h PI. Nodulation did not significantly change during the following 8 h. Dexamethasone-treated cicadas yielded significantly fewer nodules at every time point in the experiment. 3.2. Dose–response curve for dexamethasone The relationship between dexamethasone dosage and number of nodules formed in response to standard bacterial infections is presented in Fig. 3. Nodulation reactions to bacterial infections was reduced in insects treated with 10-fold increases in dexamethasone dosages. Female cicadas produce more nodules than males in response to similar bacterial challenges. For females, nodulation declined in a linear way from about 130 nodules/cicada in ethanol-treated control cicadas to about 40 nodules/cicada in females treated with the highest dexamethasone dosage. A similar pattern emerged for males, albeit at a lower range of nodules. 3.3. Influence of other eicosanoid biosynthesis inhibitors on nodulation We assessed the influence of several eicosanoid biosynthesis inhibitors on nodulation in response to infections with standard dosages of bacteria. Adult cicadas were treated with standard dosages of four inhibitors. Compared to control cicadas, the nodulation response to bacterial infections was significantly reduced
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3.4. Fatty acid rescue experiment
Fig. 3. Dose response curve for the influence of dexamethasone on nodule formation. Adults were first injected with the indicated doses of dexamethasone and then intrahemocoelically infected with approximately 1×106 cells of the bacterium S. marcescens. At 8 h PI, the test animals were anesthetized on ice, and nodulation was assessed. Each point indicates the mean number of nodules found in each insect, and the error bars represent 1 SEM (n=5 individuals for each point).
in all experimental cicada groups (Fig. 4; LSD, P⬍0.05). There were no significant differences on nodulation among animals treated with different inhibitors.
Fig. 4. Effects of treating male cicadas with individual eicosanoid biosynthesis inhibitors on nodule formation in response to intrahemocoelic infections with the insect pathogen S. marcescens. Test insects were first injected with 26 µg of either dexamethasone (Dex; PLA2 inhibitor), esculetin (Esc; lipoxygenase inhibitor), indomethacin (Indo), or naproxin (Nap; cyclooxygenase inhibitors). Control insects were first injected with ethanol (EtOH). Test and control insects were then intrahemocoelically infected with approximately 1×106 cells of the insect pathogen S. marcescens. At 8 h PI, the insects were anesthetized on ice, and nodulation was assessed. Each point indicates the mean number of nodules found in each insect, and the error bars represent 1 SEM (n=5 individuals for each point). Histogram bars with the same fill pattern are not significantly different from each other (LSD, P⬍0.05).
Dexamethasone is thought to inhibit eicosanoid biosynthesis through its effects on PLA2 (Fig. 1) and we designed experiments meant to reverse the influence of dexamethasone. Cicadas were treated with dexamethasone, then infected with bacteria. Immediately following infection, cicadas were treated with arachidonic acid. To control for the influence of the last injection on nodulation, an additional group of cicadas was injected with ethanol. The arachidonic acid treatments reversed the effects of dexamethasone on nodulation (Fig. 5; LSD, P⬍0.05). The ethanol-injected control cicadas yielded about 40 nodules/cicada and dexamethasone-treated cicadas about 20 nodules/cicada. The arachidonic acidtreated cicadas produced about 50 nodules/cicada, similar to results for control animals. The second control group, injected with a second dose of ethanol, yielded about 22 nodules/cicada. 3.5. Determining the elements of eicosanoid biosynthetic systems in fat body We recorded the presence of arachidonic acid at low proportions of tissue phospholipids from male and female cicadas using our standard chromatographic techniques. The fat body preparation from M. septendecim is competent to synthesize eicosanoids. We recorded biosynthesis of four cyclooxygenase products and a
Fig. 5. Arachidonic acid reverses the effect of dexamethasone on nodulation. Male cicadas were treated with ethanol (EtOH) or dexamethasone (Dex) and then intrahemocoelically infected with approximately 1×106 cells of the insect pathogen S. marcescens. Immediately after infection, test insects were treated with 50 µg of arachidonic acid (Dex+AA). Control insects were treated with dexamethasone and ethanol (Dex+EtOH). At 8 h PI, the insects were anesthetized on ice, and nodulation was assessed. The height of histogram bars represents the mean number of nodules found in each insect, and the error bars represent 1 SEM. The numbers in parentheses above the error bars indicate the number of insects in each category. Histogram bars with the same fill pattern are not significantly different from each other (LSD, P⬍0.05).
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Table 1 Eicosanoid biosynthesis by microsomal-enriched preparations of fat body from adult cicadas, M. septendecima Eicosanoid product
Male
Female
PGA2 PGE2 PGD2 PGF2α HETEb
0.057±0.016 0.036±0.010 0.040±0.011 0.036±0.014 0.043±0.001
0.000±0.014 0.011±0.008 0.00±0.012 0.006±0.007 0.013±0.019
a Values represent eicosanoid biosynthesis rates as pmol/mg/hr±1 SEM, n=3 separate experiments with pools of fat bodies from 5 individuals per gender. b HETE=hydroxyeicosatetraenoic acid, tentative identification.
lipoxygenase product tentatively identified as a hydroxyeicosatetraenoic acid (Table 1). We also recorded the presence of a digestive PLA2 in homogenates of whole alimentary tracts from male and female cicadas (Table 2). Males yielded substantial enzyme activity, about 22 pmol/min/mg protein. Virtually no activity was detected in female alimentary tracts.
4. Discussion In this paper we report the outcomes of testing the eicosanoid hypothesis in exopterygote insects featuring an unusual life history: namely, adults of the cicada, M. septendecim and M. cassini. Several lines of evidence presented here support the hypothesis. First, dexamethasone treatments significantly reduced nodulation throughout the time course of the experiments. Second, the influence of dexamethasone on nodulation was expressed in a dose-dependent manner. Third, independent inhibition of the two major eicosanoid biosynthetic pathways, cyclooxygenase and lipoxygenase, significantly reduced nodulation in infected cicadas. Fourth, the influence of dexamethasone on nodulation was reversed by treating infected cicadas with arachidonic acid, an eicosanoid-precursor polyunsaturated fatty acid. Fifth, we recorded the presence of a digestive PLA2 which can hydrolyze arachidonic acid from phospholipids. Finally, we determined the presence arachidonic acid Table 2 Digestive phospholipase A2 activity in homogenates of complete alimentary tracts of adult periodical cicadas, M. septendecima Gender
PLA2 Activity
Male Female
22.3±1.50 0.3±0.07
a Values represent enzyme activity as pmol hydrolyzed arachidonate/min/mg protein, ±1 SEM, n=3 experiments with pools of 6 alimentary tracts per gender.
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and of eicosanoid biosynthesizing enzymes in fat body of adult male and female cicadas. These results agree with our original findings with the tobacco hornworm, M. sexta (Miller et al., 1994), as well as with outcomes of experiments on other endopterygote insect species (Miller et al., 1996; Jurenka et al., 1997; StanleySamuelson et al., 1997). We reported similar findings with an orthopteroid insect, adults of the cricket G. assimilis (Miller et al., 1998). The significance of the information reported in this paper lies in extending the eicosanoid hypothesis to two hemipteroid insect species with very long life history patterns. These findings support our broader hypothesis that eicosanoids mediate nodulation and other cellular immune reactions in most, if not all, insects. We determined the cellular immune reactions to bacterial infections in G. assimilis are similar in males and females (Miller et al., 1998). The situation is considerably different in cicadas. As seen from the dose– response data, females produced far more nodules than males in response to similar bacterial challenges. Similarly, our time course experiments show that adult male cicadas produce a maximum of about 45–55 nodules/individual, while females produce over 100 nodules/individual. As discussed elsewhere (Miller et al., 1998), differences in nodulation might be due to differences in circulating hemocyte populations (Miller et al., 1996; Howard et al., 1998). If this is so, we would suppose female cicadas have more circulating hemocytes than males. While easily counted in samples from many insect species, it is difficult to assess hemocyte populations in cicadas because they have very little circulating hemolymph (Brown and Chippendale, 1973). Nonetheless, if the situation is otherwise, then other factors, not yet illuminated, influence insect capacities to form nodules. Nodulation intensity in male cicadas is similar to nodulation intensity in larvae of the tenebrionid beetle, Z. atratus (Miller et al., 1996), and is considerably lower than the hundred or more nodules/individual seen in tobacco hornworms (Miller et al., 1994). In identical experiments with silkworms, we recorded about 80 nodules/individual (Stanley-Samuelson et al., 1997). It is clear that insect species differ in their expression of nodulation reactions to similar bacterial challenges. Turning to another gender difference in cicadas, we also note that males yielded about 100-fold more digestive PLA2 activity than females. PLA2 activity in the male cicada preparations (about 22 pmol/min/mg protein) was 2 to 3 times lower than the activity we recorded in similar preparations from two other species, tobacco hornworms, M. sexta (Rana et al., 1998) and mosquito larvae, Aedes aegypti (Nor Aliza and Stanley, 1998). The higher levels of digestive PLA2 activity in male cicadas, relative to females, may be indicative of more active feeding by males to support their energeti-
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cally expensive calling behavior. Recording low levels of PLA2 activity in female cicadas is consistent with our findings with tobacco hornworms and mosquito larvae, in which fasting individuals yielded much reduced PLA2 activity. The significance of assessing the presence of a digestive PLA2 in adult cicadas is indirectly connected to the biological roles of eicosanoids in these insects. As we have discussed for other species, the digestive PLA2 is an essential element of digestive physiology, responsible for hydrolyzing polyunsaturated fatty acids from dietary phospholipids to facilitate uptake of the free fatty acids into the cicada body. The presence of substantial digestive PLA2 activity in midguts of male cicadas indicates that dietary polyunsaturated fatty acids are important in the nutrition or energetics of these males. These dietary components are presumably important in eicosanoid biosynthesis, as well as several other aspects of physiology (Dadd, 1985). The dexamethasone dose–response curve and the results of the arachidonic acid rescue experiments strongly support the eicosanoid hypothesis. Moreover, a variety of eicosanoid biosynthesis inhibitors, specific for cyclooxygenase and lipoxygenase pathways, effectively retarded nodulation in adult cicadas. This apparent involvement of both cyclooxygenase and lipoxygenase products is probably due to the complexity of nodule formation, which involves many separate cellular actions, the inhibition of which may impact the overall nodulation process. Recognition of the biological significance of eicosanoids in insects and other invertebrates is rapidly increasing (Stanley-Samuelson, 1994a,b; Stanley-Samuelson and Pedibhotla, 1996; Stanley and Howard, 1998). Nonetheless, we have argued that the biochemistry of eicosanoids in insects is understudied (Miller et al., 1998). The presence of eicosanoid-precursor polyunsaturated fatty acids and eicosanoid biosynthesizing enzymes in insect tissues is a crucial point in discussions of the roles of eicosanoids in any aspect of insect physiology. In this work on cicadas, we used standard protocols to record the presence of arachidonic acid in cicada tissue phospholipids. As we observed with the desert cicada, Tibican dealbatus, the arachidonic acid was present in very low proportions of tissue phospholipids (Stanley-Samuelson et al., 1990). We also demonstrated that fat body preparations from M. septendecim are competent to biosynthesize eicosanoids from exogenous radioactive arachidonic acid. There is considerable variation in rates of prostaglandin biosynthesis among invertebrates. Total prostaglandin biosynthesis in the fat body preparations was on par with our findings with the desert cicada, T. dealbatus (Stanley-Samuelson et al., 1990), with fat body preparations from the silkmoth, B. mori, and with whole animal preparations of the lone star tick (Pedibhotla et al., 1995). On the other hand, the rates we observed with M. septendecim were lower than pro-
staglandin biosynthesis rates in tobacco hornworm hemocytes by approximately an order of magnitude (Gadelhak et al., 1995). Fat body is an immunity-conferring tissue in insects, and at least in the silkmoth fat body prostaglandins are thought to mediate induction of two genes for antibacterial proteins (Morishima et al., 1997). We note this work with cicada fat body does not directly demonstrate that cicada hemocytes are competent to produce eicosanoids. Nevertheless, these results indicate a tissue capacity for eicosanoid biosynthesis in cicadas, and we take these findings to support the eicosanoid hypothesis.
Acknowledgements We thank Dr Ralph Howard and Dr E.A. Heinrichs for reading and providing useful comments on a draft of this paper. This is paper no. 12,335 of the Nebraska Agricultural Research Division, and contribution no. 1,000 of the Department of Entomology. This work was supported by the Agricultural Research Division, UNL (Project NEB-17-054) and by USDA-ARS Specific Cooperative Agreement # 58-5430-5-115.
References Beckage, N.E., Thompson, S.N., Federici, B.A. (Eds.), 1993. Parasites and Pathogens of Insects, vol. 2: Pathogens. Academic Press, New York. Brown, J.J., Chippendale, G.M., 1973. Nature and fate of nutrient reserves of the periodical (17-year) cicada. Journal of Insect Physiology 19, 607–614. Dadd, R.H., 1985. Nutrition: organisms. In: Kerkut, G.A., Gilbert, L.I. (Eds.), Comprehensive Insect Physiology, Biochemistry and Pharmacology, vol. 4. Pergamon Press, Oxford, pp. 313–390. Daly, H.V., Doyen, J.T., Purcell III, A.H., 1998. Introduction to Insect Biology and Diversity. 2nd ed. Oxford University Press, Oxford. Gadelhak, G.G., Pedibhotla, V.K., Stanley-Samuelson, D.W., 1995. Eicosanoid biosynthesis by hemocytes from the tobacco hornworm, Manduca sexta. Insect Biochemistry and Molecular Biology 25, 743–749. Gupta, A.P. (Ed.), 1991. Immunology of Insects and Other Arthropods. CRC Press, Boca Raton, FL. Horohov, D.W., Dunn, P.E., 1983. Phagocytosis and nodule formation by hemocytes of Manduca sexta following injection of Pseudomonas aeruginosa. Journal of Invertebrate Pathology 41, 203–213. Howard, R.W., Miller, J.S., Stanley, D.W., 1998. The influence of bacterial species and intensity of infections on nodule formation in insects. Journal of Insect Physiology 44, 157–164. Jurenka, R.A., Miller, J.S., Pedibhotla, V.K., Rana, R.L., StanleySamuelson, D.W., 1997. Eicosanoids mediate microaggregation and nodulation responses to bacterial infections in black cutworms, Agrotis ipsilon, and true armyworms, Pseudaletia unipuncta. Journal of Insect Physiology 43, 125–133. Mandato, C.A., Diehl-Jones, W.L., Moore, S.J., Downer, R.G.H., 1997. The effects of eicosanoid biosynthesis inhibitors on prophenoloxidase activation, phagocytosis and cell spreading in Galleria mellonella. Journal of Insect Physiology 43, 1–8. Miller, J.S., Nguyen, T., Stanley-Samuelson, D.W., 1994. Eicosanoids
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mediate insect nodulation responses to bacterial infections. Proceedings of the National Academy of Sciences USA 91, 12418– 12422. Miller, J.S., Howard, R.W., Nguyen, T., Nguyen, A., Rosario, R.M.T., Stanley-Samuelson, D.W., 1996. Eicosanoids mediate nodulation responses to bacterial infections in larvae of the tenebrionid beetle, Zophobas atratus. Journal of Insect Physiology 42, 3–12. Miller, J.S., Howard, R.W., Rana, R.L., Tunaz, H., Stanley, D.W., 1999. Eicosanoids mediate nodulation reactions to bacterial infections in adults of the cricket, Gryllus assimilis. Journal of Insect Physiology 45, 75–83. Moore, T.E., Alexander, R.D., 1958. The periodical cicada complex (Homoptera: Cicadidae). In: Proceedings of the Tenth International Congress of Entomology, vol. 1, pp. 349–356. Morishima, I., Yamano, Y., Inoue, K., Matsuo, N., 1997. Eicosanoids mediate induction of immune genes in the fat body of the silkworm, Bombyx mori. Federation of European Biochemical Societies Letters 419, 83–86. Nor Aliza, A.R., Stanley, D.W., 1998. A digestive phospholipase A2 in larval mosquitoes, Aedes aegypti. Insect Biochemistry and Molecular Biology 28, 561–569. Pedibhotla, V.K., Sarath, G., Sauer, J.R., Stanley-Samuelson, D.W., 1995. Prostaglandin biosynthesis and subcellular localization of prostaglandin H synthase activity in the lone star tick, Amblyomma americanum. Insect Biochemistry and Molecular Biology 25, 1027–1039. Rana, R.L., Sarath, G., Stanley, D.W., 1998. A digestive phospholipase A2 in midguts of tobacco hornworms, Manduca sexta L. Journal of Insect Physiology 44, 297–303. Stanley, D.W., 1999. Eicosanoids in Invertebrate Signal Transduction Systems (in press). Princeton University Press, Princeton.
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Stanley, D.W., Howard, R.W., 1998. The biology of prostaglandins and related eicosanoids in invertebrates: cellular, organismal and ecological actions. American Zoologist 38, 369–381. Stanley-Samuelson, D.W., 1994a. Assessing the significance of prostaglandins and other eicosanoids in insect physiology. Journal of Insect Physiology 40, 3–11. Stanley-Samuelson, D.W., 1994b. Prostaglandins and related eicosanoids in insects. Advances in Insect Physiology 24, 115–212. Stanley-Samuelson, D.W., Ogg, C.L., 1994. Prostaglandin biosynthesis by fat body from the tobacco hornworm, Manduca sexta. Insect Biochemistry and Molecular Biology 24, 481–491. Stanley-Samuelson, D.W., Pedibhotla, V.K., 1996. What can we learn from prostaglandins and related eicosanoids in insects? Insect Biochemistry and Molecular Biology 26, 223–234. Stanley-Samuelson, D.W., Howard, R.W., Toolson, E.C., 1990. Phospholipid fatty acid composition and arachidonic acid uptake and metabolism by the cicada Tibicen dealbatus (Homoptera: Cicadidae). Comparative Biochemistry and Physiology 97B, 285–289. Stanley-Samuelson, D.W., Pedibhotla, V.K., Rana, R.L., Nor Aliza, A.R., Hoback, W.W., Miller, J.S., 1997. Eicosanoids mediate nodulation responses to bacterial infections in larvae of the silkmoth, Bombyx mori. Comparative Biochemistry and Physiology 118A, 93–100. Toolson, E.C., Ashby, P., Howard, R.W., Stanley-Samuelson, D.W., 1994. Eicosanoids mediate control of thermoregulatory sweating in the cicada, Tibicen dealbatus (Insecta: Homoptera). Journal of Comparative Physiology B 164, 278–285. Wiesner, A., Dunphy, G.B., Marmaras, V.J., Morishima, I., Sugumaran, M., Yamakawa, M. (Eds.), 1998. Techniques in Insect Immunology. SOS Publications, Fair Haven, NJ.
Eicosanoids mediate nodulation reactions to bacterial infections in adults of two 17-year periodical cicadas, Magicicada septendecim and M. cassini Hasan Tunaz, Jon C. Bedick, Jon S. Miller, W. Wyatt Hoback, Rico L. Rana, David W. Stanley * Insect Biochemical Physiology Laboratory, University of Nebraska, Lincoln, NE 68583-0816, USA Received 17 August 1998; accepted 11 December 1998
Abstract 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 A2, 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. 1999 Elsevier Science Ltd. All rights reserved. Keywords: Cellular immunity; Eicosanoid biosynthesis; Hemiptera; Phospholipase A2
1. Introduction We have been investigating the idea that eicosanoids mediate cellular defense reactions, particularly nodulation, to bacterial infections in insects. Eicosanoid is a collective term for all oxygenated metabolites of three C20 polyunsaturated fatty acids, specifically 20:3n-6, 20:4n-6 and 20:5n-3. Three main groups of eicosanoids are recognized (Fig. 1; Stanley-Samuelson, 1994a). Prostaglandins and thromboxanes are products of the cyclooxygenase pathways. Epoxyeicosatrienoic acids are products of cytochrome P450s, often called epoxygenase pathways for convenience. The many lipoxygenase systems yield a complex assemblage of products, including
* Corresponding author. Tel.: +1-402-472-8710; fax: +1-402-4728746. E-mail address: DStanley얀unlnotes.unl.edu (D.W. Stanley)
leukotrienes. Various eicosanoids are present and biologically active in virtually all mammalian tissues and body fluids. Eicosanoids also exert many actions in invertebrates, including aspects of reproduction, ion transport, and host–parasite relationships, all detailed in recent reviews (Stanley-Samuelson, 1994a,b; Stanley and Howard, 1998; Stanley, 1999). Nodulation is a cellular reaction which clears large numbers of bacterial cells from circulation during the early phase of an infection in tobacco hornworms, typically the first 2 hours (Horohov and Dunn, 1983). Nodulation is a complex process beginning with microaggregations of hemocytes (Gupta, 1991; Beckage et al., 1993; Wiesner et al., 1998). Bacterial cells are entrapped within the microaggregates. The microaggregates continue to grow by attracting hemocytes (presumably by chemotaxis, as seen in mammalian systems) and attached bacteria. The process ends with a phase of melanization, which leaves darkened nodules, typically about 0.1 mm
0022-1910/99/$ - see front matter. 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 2 - 1 9 1 0 ( 9 9 ) 0 0 0 7 2 - 4
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Fig. 1. An outline of eicosanoid biosynthesis as understood from research on mammalian systems. Three C20 polyunsaturated fatty acids, 20:3n6, 20:4n-6 and 20:5n-3 are potential substrates for eicosanoid biosynthesis, of which metabolism of arachidonic acid is best understood. Chemical structures are denoted by numerals and major biosynthetic pathways are indicated by capital letters. 1=a cellular phospholipid. 2=hydrolyzed arachidonic acid. 3=prostaglandin E2. 4=5-hydroperoxyeicosatetraenoic acid. 5=leukotriene B4. 6=11,12-epoxyeicosatrienoic acid. 7=lipoxin A. A=phospholipase A2; B=cyclooxygenase and associated enzyme steps; C=cytochrome P450 epoxygenase; D=lipoxygenase.
in diameter, attached to various organs and body walls. Hence, bacterial cells are cleared from circulation in a topological sense by mass entrapments. We tested the idea that eicosanoids mediate nodulation reactions to bacterial infections in tobacco hornworms (Miller et al., 1994). Outcomes of these experiments showed that inhibition of eicosanoid biosynthesis immediately prior to bacterial infections severely impaired the ability of the hornworms to form microaggregates and nodules in response to bacterial infections.
Using similar experimental protocols, we have shown that microaggregation and nodulation reactions to bacterial infections depend on eicosanoid biosynthesis in the tenebrionid beetle, Zophobas atratus (Miller et al., 1996), in the silkworm, Bombyx mori (Stanley-Samuelson et al., 1997), and in two other moths, black cutworms Agrotis ipsilon and true armyworms Pseudaletia unipuncta (Jurenka et al., 1997). Mandato et al. (1997) showed that cell spreading, a distinct phase of nodulation, prophenyloxidase activation, and phagocytosis are mediated by eicosanoids in waxmoths, Galleria mel-
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lonella. All experiments have so far produced results supporting the idea that eicosanoids mediate nodulation reactions to bacterial challenges in insects, which we now formalize as the eicosanoid hypothesis. Most recently we tested the hypothesis in an exopterygote insect, adults of the cricket Gryllus assimilis (Miller et al., 1999). Taken together, these findings strongly support the eicosanoid hypothesis. Nevertheless, as a single species taken from a rather large assemblage of insects, it is difficult to extrapolate from the cricket G. assimilis to the remaining orders within the Division Exopterygota. The extrapolation is more speculative because the orders within this Division are phylogenetically diverse (Daly et al., 1998). The cricket is an orthopteran grouped within the Superorder Orthopteroidea. The other main Superorder is the Hemipteroidea, a large monophyletic group completely distinct from the Orthopteroidea (Daly et al., 1998). We recognize that the eicosanoid hypothesis is only weakly supported by studies of a single orthopteroid insect species. Hence, we deemed it necessary to test the eicosanoid hypothesis in a representative of the Hemipteroidea. The biology of hemipteroids differs in important ways from all other insects we have tested so far. Hemipteriod species are fluid-feeding insects during all post-embryonic life stages, and they exhibit many unusual life history patterns. One group of hemipteroids, the periodical cicadas are remarkably specialized insects which spend extended larval periods underground and short adult phases above ground. Another good reason to select cicadas as an experimental insect is that eicosanoids have been implicated in another area of cicada physiology. Toolson et al. (1994) suggested that eicosanoids regulate the thermoregulatory set points in the desert cicada, Tibicen dealbatus. If this is so for other cicadas, including periodical cicadas, we can point to the possibility of two distinct roles of eicosanoids in cicadas: one in thermobiology and the second in immunity. Multiple eicosanoid actions are commonly recognized in mammals, and have been postulated for silkmoths, B. mori. Prostaglandins release egg-laying behavior, modulate release of lipids from fat body and mediate nodulation reactions to infections in this species (Stanley-Samuelson et al., 1997). As seen in mammals, invertebrates probably rely on eicosanoid actions that operate independently in many different physiological systems within the same organism. Hence, adult periodical cicadas present an opportunity to test the hypothesis that eicosanoids mediate cellular immune reactions in the Hemipteriodea. In this paper, we report that eicosanoids mediate nodulation reactions to bacterial infections in adults of two hemipteran insects, the 17-year periodical cicadas, Magicicada septendecim and M. cassini. We show that adults of these species form nodules in response to bacterial infections, and that the nodulation response is sev-
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erely impaired by treating the cicadas with eicosanoid biosynthesis inhibitors prior to infection.
2. Materials and methods 2.1. Organisms We collected adult cicadas, M. septendecim and M. cassini within one week of adult emergence from Schramm State Park, in eastern Nebraska. After identifying males and females to species on morphological grounds (Moore and Alexander, 1958), we held them overnight in a laboratory refrigerator. The insects were removed to room temperature and allowed to recover normal activity before experimental manipulations. Cultures of a nonpigmented strain of Serratia marcescens and nutrient broth (Difco) were purchased from Carolina Biological Supply (Burlington, NC). Bacteria were grown in 50 ml of nutrient broth in an environmental shaker at 37°C and 100 rpm. Bacteria were grown to a titre of 107 colony forming units (cfu)/ml, and used in stationary phase. 2.2. Injections and assays for nodulation Test insects were injected with either the PLA2 inhibitor dexamethasone [(11β, 16α)-9-fluoro-11,17,21-trihydroxy-16-methylpregna-1,4-dione], one of the cyclooxygenase inhibitors indomethacin [1-p-chlorobenzoyl)-5methoxy-2-methyl-3-indolyl-acetic acid], naproxin [D-2(6-methoxy-2-naphthyl)propionic acid], or the 5- and 12lipoxygenase inhibitor esculetin [6,7-dihydroxycoumarin] (all inhibitors from BioMol, Plymouth Meeting, PA). In some experiments, cicadas were also injected with arachidonic acid [5,8,11,14-eicosatetraenoic acid] (Sigma Chemical Co., St. Louis, MO). Control cicadas were injected with nutrient broth, or 95% ethanol. All injections of pharmaceuticals were in a standard dosage of 26 µg in 10 µl of ethanol, except in dose–response experiments. The fatty acids were injected at dosages of 50 µg in 5 µl of ethanol per adult cicada. Prior to injections, cicadas were surface sterilized by swabbing their surfaces with 95% ethanol. Insects were infected by injecting a bacterial dosage of 106 cfu/cicada (Miller et al., 1994, 1996) in 100 µl aliquots using a 26 gauge 0.5⬙ needle attached to a 1000 µl syringe. Drugs and control substances were injected into the opposite lateral side of the abdomen in 10 µl volumes using a 10 µl Hamilton 701 syringe. Nodulation was assessed at selected times post-infection (PI). Cicadas were anesthetized by chilling on ice, then the hemocoels were exposed. Melanized, dark nodules were counted under a stereomicroscope at 60×. The nodules were distinct, and counting reliably reflected the extent on the nodulation response to infections (Miller
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et al., 1994, 1996). The gonads, fat bodies and other internal tissues were carefully probed for previously unseen nodules. We considered the possibility that nodulation reactions to bacterial infections in M. septendecim and M. cassini might be quantitatively different. After routine bacterial challenges, both species formed similar numbers of nodules, 35±4.4/individual for M. septendecim males and 38±3 nodules/individual for M. cassini males (n=5 for each species, not significantly different by Student’s t test). We used males of the larger M. septendecim for most manipulations, but approximately 10% of the individuals were males of M. cassini. The data for both species were combined for presentation, and the implications of the experimental results apply to both species. 2.3. Time-course of nodulation: influence of dexamethasone Individuals in two groups of male cicadas were injected with 10 µl of ethanol or with 26 µg of dexamethasone in 10 µl of ethanol. The cicadas were immediately injected with bacteria as described. At 2, 4, 8, and 16 h PI, sub-groups of control and experimental insects were anesthetized and nodulation assessed. 2.4. Dose–response curve for dexamethasone We conducted two separate experiments, one with males and one with females. Individuals in four groups of cicadas were injected with 10 µl of ethanol, or with 0.26, 2.6 or 26 µg of dexamethasone in 10 µl of ethanol, then injected with a standard dosage of bacteria. At 8 h PI, the cicadas were anesthetized, and nodulation was assessed. Males and females differ in internal anatomy. Most of the abdomen of males is as a hollow resonance chamber, while the abdomens of females are filled with fat body and well-developed ovaries. Because of these anatomical differences we used male cicadas in all remaining experiments because the differences facilitated assessment of nodulation. 2.5. Fatty acid rescue experiments Individuals in two groups of cicadas were injected with either 10 µl of ethanol or 26 µg of dexamethasone in 10 µl of ethanol, then injected with bacteria as described. Immediately after infection, the dexamethasone-treated cicadas were divided into two subgroups. Individuals in one sub-group were treated with 50 µg of arachidonic acid in 5 µl of ethanol. Individuals in another sub-group were treated with 5 µl of ethanol to control for the effects of the extra injection on nodulation. At 8 h PI, the cicadas were anesthetized and nodulation was assessed.
2.6. Influence of other eicosanoid biosynthesis inhibitors on nodulation Cicadas were divided into groups and injected with either one of the cyclooxygenase inhibitors indomethacin or naproxin, or the lipoxygenase inhibitor esculetin, all in 10 µl of ethanol. Control insects were injected with 10 µl of ethanol. Following injections, the cicadas were infected with a standard dosage of bacteria as described. At 8 h PI, the cicadas were anesthetized and nodulation was assessed. 2.7. Eicosanoid biosynthesis by cicada fat body preparations We investigated prostaglandin (PG) biosynthesis by microsomal-enriched preparations of fat body from adult cicadas. These experiments followed protocols developed for fat body from M. sexta (Stanley-Samuelson and Ogg, 1994). Briefly, fat body was removed from chilled adult male and female cicadas, then homogenized in a glass homogenizer. The homogenates were sonicated for 10 s at 40 W using a VibraCell sonicator (VibraCell, Danbury, CT). This preparation was centrifuged for 10 min at 735g, and the supernatant was centrifuged for another 20 min at 16,000g, both steps at 4°C. The 16,000g microsomal-enriched supernatants were used in all experiments. Protein concentrations in these preparations were determined against bovine serum albumin using the bicinchoninic acid reagent (Pierce, Rockford, IL). Standard curves and samples were read on a BioTek microtitre plate reader at 562 nm. Radioactive arachidonic acid (5,6,8,9,11,12,14,15-3H20:4, 60–100 Ci/mmol) was purchased from DuPont. The incubation buffer was 0.05 M KH2PO4, pH 8.0, amended with a standard co-factor mixture (2.4 mM reduced glutathione, 0.25 mM hydroquinone and 25 µg hemoglobin). For each PG biosynthesis reaction, 0.4 µCi of labeled arachidonic acid was dispensed into reaction tubes and the solvent was evaporated. The reactions were carried out in 1.0 ml total volume. The experiments were preceded by a 10 min pre-incubation with all reaction components, except the protein source. The protein source was added (1.5 mg/ml in each reaction), and after a 2 min reaction period, the reactions were stopped by addition of 500 µl 0.1 N HCl. Reaction products were extracted from the acidified reaction mixture three times in ethyl acetate. The combined extracts, containing PGs and possibly lipoxygenase products, were evaporated under N2. A mixture of appropriate eicosanoid standards was added to each sample, then samples were applied to thin-layer chromatography plates. The plates were developed and fractions observed as described previously (Stanley-Samuelson and Ogg, 1994). Bands corresponding in Rf to selected authentic eicosanoid standards and to free fatty acids were transferred to liquid
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scintillation vials. Radioactivity in each fraction was determined by adding Ecolite scintillation mixture (ICN Biomedicals, Irvine, CA) and counting on a LKB Wallac 1209 Rackbeta Liquid Scintillation Counter (Pharmacia, Turku, Finland) at 50% counting efficiency for 3H. Eicosanoid biosynthesis was calculated from the liquid scintillation data. In negative control experiments, microsomal-enriched preparations were heated in boiling water for 15 min before the experiments, and processed as just described. The results of these control experiments were used to correct values from biosynthesis experiments as described (Stanley-Samuelson and Ogg, 1994). 2.8. Assessing digestive phospholipase A2 in midgut contents We used standard procedures to assess digestive PLA2 activity (Rana et al., 1998; Nor Aliza and Stanley, 1998). Digestive tracts were dissected from males and females of M. septidecim, homogenized in buffer (0.1 M Tris, 5 mM CaCl2, pH 9.0), then centrifuged at 11,750g for 10 min. The resulting supernatants were used as buffer-soluble enzyme preparations, which were held on ice until further use. Protein concentrations in the supernatants were determined as described in the preceding section. Radioactive phosphatidylcholine (1-palmitoyl, 2-arachidonyl [arachdonyl-1-14C]; 1.9 GBq/mmol; New England Nuclear, Wilmington, DE) was prepared in vesicles by vigorously vortexing in buffer. The final radioactive substrate concentration was 0.25 µCi/250 µl reaction volume. The PLA2 reactions were initiated by adding aliquots of enzyme source (2.5 mg protein) to tubes containing substrate vesicles. After 30 min incubations at 28°C, the reactions were terminated by adding 0.5 ml of chloroform:methanol, 2:1, v/v, acidified with 50 µl 2 M HCl. Cold arachidonic acid was added to serve as a carrier. Two additional extraction steps using 0.5 ml chloroform followed. Free fatty acids were isolated by TLC plates developed in petroleum ether:ethyl ether:acetic acid (80:20:1, v/v). Fractions corresponding to free fatty acids and to phospholipids were transferred to liquid scintillation vials. Radioactivity in each vial was determined as described above. 2.9. Statistical analysis Data on nodulation were analyzed by ANOVA and significance was determined at P⬍0.05 using the Least Significant Difference (LSD) test. 3. Results 3.1. Time course of nodulation The time course of visible nodule formation in two groups of cicadas is displayed in Fig. 2. The ethanol-
Fig. 2. Time course of nodulation in adult male cicadas in response to intrahemocoelic infections with the pathogenic bacterium S. marcescens. Test insects were first injected with dexamethasone (unfilled circles), and control insects were first injected with ethanol (filled circles). Within 3–10 min, both groups of insects were then intrahemocoelically infected with approximately 1×106 bacterial cells. At the indicated times PI, the insects were anesthetized on ice and nodulation assessed. Each point indicates the mean number of nodules found in each insect, and the error bars represent 1 SEM (n=5 individuals for each point).
treated control insects produced about 20 nodules/cicada at 2 h PI, which increased to nearly 60 nodules/cicada by 8 h PI. Nodulation did not significantly change during the following 8 h. Dexamethasone-treated cicadas yielded significantly fewer nodules at every time point in the experiment. 3.2. Dose–response curve for dexamethasone The relationship between dexamethasone dosage and number of nodules formed in response to standard bacterial infections is presented in Fig. 3. Nodulation reactions to bacterial infections was reduced in insects treated with 10-fold increases in dexamethasone dosages. Female cicadas produce more nodules than males in response to similar bacterial challenges. For females, nodulation declined in a linear way from about 130 nodules/cicada in ethanol-treated control cicadas to about 40 nodules/cicada in females treated with the highest dexamethasone dosage. A similar pattern emerged for males, albeit at a lower range of nodules. 3.3. Influence of other eicosanoid biosynthesis inhibitors on nodulation We assessed the influence of several eicosanoid biosynthesis inhibitors on nodulation in response to infections with standard dosages of bacteria. Adult cicadas were treated with standard dosages of four inhibitors. Compared to control cicadas, the nodulation response to bacterial infections was significantly reduced
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3.4. Fatty acid rescue experiment
Fig. 3. Dose response curve for the influence of dexamethasone on nodule formation. Adults were first injected with the indicated doses of dexamethasone and then intrahemocoelically infected with approximately 1×106 cells of the bacterium S. marcescens. At 8 h PI, the test animals were anesthetized on ice, and nodulation was assessed. Each point indicates the mean number of nodules found in each insect, and the error bars represent 1 SEM (n=5 individuals for each point).
in all experimental cicada groups (Fig. 4; LSD, P⬍0.05). There were no significant differences on nodulation among animals treated with different inhibitors.
Fig. 4. Effects of treating male cicadas with individual eicosanoid biosynthesis inhibitors on nodule formation in response to intrahemocoelic infections with the insect pathogen S. marcescens. Test insects were first injected with 26 µg of either dexamethasone (Dex; PLA2 inhibitor), esculetin (Esc; lipoxygenase inhibitor), indomethacin (Indo), or naproxin (Nap; cyclooxygenase inhibitors). Control insects were first injected with ethanol (EtOH). Test and control insects were then intrahemocoelically infected with approximately 1×106 cells of the insect pathogen S. marcescens. At 8 h PI, the insects were anesthetized on ice, and nodulation was assessed. Each point indicates the mean number of nodules found in each insect, and the error bars represent 1 SEM (n=5 individuals for each point). Histogram bars with the same fill pattern are not significantly different from each other (LSD, P⬍0.05).
Dexamethasone is thought to inhibit eicosanoid biosynthesis through its effects on PLA2 (Fig. 1) and we designed experiments meant to reverse the influence of dexamethasone. Cicadas were treated with dexamethasone, then infected with bacteria. Immediately following infection, cicadas were treated with arachidonic acid. To control for the influence of the last injection on nodulation, an additional group of cicadas was injected with ethanol. The arachidonic acid treatments reversed the effects of dexamethasone on nodulation (Fig. 5; LSD, P⬍0.05). The ethanol-injected control cicadas yielded about 40 nodules/cicada and dexamethasone-treated cicadas about 20 nodules/cicada. The arachidonic acidtreated cicadas produced about 50 nodules/cicada, similar to results for control animals. The second control group, injected with a second dose of ethanol, yielded about 22 nodules/cicada. 3.5. Determining the elements of eicosanoid biosynthetic systems in fat body We recorded the presence of arachidonic acid at low proportions of tissue phospholipids from male and female cicadas using our standard chromatographic techniques. The fat body preparation from M. septendecim is competent to synthesize eicosanoids. We recorded biosynthesis of four cyclooxygenase products and a
Fig. 5. Arachidonic acid reverses the effect of dexamethasone on nodulation. Male cicadas were treated with ethanol (EtOH) or dexamethasone (Dex) and then intrahemocoelically infected with approximately 1×106 cells of the insect pathogen S. marcescens. Immediately after infection, test insects were treated with 50 µg of arachidonic acid (Dex+AA). Control insects were treated with dexamethasone and ethanol (Dex+EtOH). At 8 h PI, the insects were anesthetized on ice, and nodulation was assessed. The height of histogram bars represents the mean number of nodules found in each insect, and the error bars represent 1 SEM. The numbers in parentheses above the error bars indicate the number of insects in each category. Histogram bars with the same fill pattern are not significantly different from each other (LSD, P⬍0.05).
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Table 1 Eicosanoid biosynthesis by microsomal-enriched preparations of fat body from adult cicadas, M. septendecima Eicosanoid product
Male
Female
PGA2 PGE2 PGD2 PGF2α HETEb
0.057±0.016 0.036±0.010 0.040±0.011 0.036±0.014 0.043±0.001
0.000±0.014 0.011±0.008 0.00±0.012 0.006±0.007 0.013±0.019
a Values represent eicosanoid biosynthesis rates as pmol/mg/hr±1 SEM, n=3 separate experiments with pools of fat bodies from 5 individuals per gender. b HETE=hydroxyeicosatetraenoic acid, tentative identification.
lipoxygenase product tentatively identified as a hydroxyeicosatetraenoic acid (Table 1). We also recorded the presence of a digestive PLA2 in homogenates of whole alimentary tracts from male and female cicadas (Table 2). Males yielded substantial enzyme activity, about 22 pmol/min/mg protein. Virtually no activity was detected in female alimentary tracts.
4. Discussion In this paper we report the outcomes of testing the eicosanoid hypothesis in exopterygote insects featuring an unusual life history: namely, adults of the cicada, M. septendecim and M. cassini. Several lines of evidence presented here support the hypothesis. First, dexamethasone treatments significantly reduced nodulation throughout the time course of the experiments. Second, the influence of dexamethasone on nodulation was expressed in a dose-dependent manner. Third, independent inhibition of the two major eicosanoid biosynthetic pathways, cyclooxygenase and lipoxygenase, significantly reduced nodulation in infected cicadas. Fourth, the influence of dexamethasone on nodulation was reversed by treating infected cicadas with arachidonic acid, an eicosanoid-precursor polyunsaturated fatty acid. Fifth, we recorded the presence of a digestive PLA2 which can hydrolyze arachidonic acid from phospholipids. Finally, we determined the presence arachidonic acid Table 2 Digestive phospholipase A2 activity in homogenates of complete alimentary tracts of adult periodical cicadas, M. septendecima Gender
PLA2 Activity
Male Female
22.3±1.50 0.3±0.07
a Values represent enzyme activity as pmol hydrolyzed arachidonate/min/mg protein, ±1 SEM, n=3 experiments with pools of 6 alimentary tracts per gender.
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and of eicosanoid biosynthesizing enzymes in fat body of adult male and female cicadas. These results agree with our original findings with the tobacco hornworm, M. sexta (Miller et al., 1994), as well as with outcomes of experiments on other endopterygote insect species (Miller et al., 1996; Jurenka et al., 1997; StanleySamuelson et al., 1997). We reported similar findings with an orthopteroid insect, adults of the cricket G. assimilis (Miller et al., 1998). The significance of the information reported in this paper lies in extending the eicosanoid hypothesis to two hemipteroid insect species with very long life history patterns. These findings support our broader hypothesis that eicosanoids mediate nodulation and other cellular immune reactions in most, if not all, insects. We determined the cellular immune reactions to bacterial infections in G. assimilis are similar in males and females (Miller et al., 1998). The situation is considerably different in cicadas. As seen from the dose– response data, females produced far more nodules than males in response to similar bacterial challenges. Similarly, our time course experiments show that adult male cicadas produce a maximum of about 45–55 nodules/individual, while females produce over 100 nodules/individual. As discussed elsewhere (Miller et al., 1998), differences in nodulation might be due to differences in circulating hemocyte populations (Miller et al., 1996; Howard et al., 1998). If this is so, we would suppose female cicadas have more circulating hemocytes than males. While easily counted in samples from many insect species, it is difficult to assess hemocyte populations in cicadas because they have very little circulating hemolymph (Brown and Chippendale, 1973). Nonetheless, if the situation is otherwise, then other factors, not yet illuminated, influence insect capacities to form nodules. Nodulation intensity in male cicadas is similar to nodulation intensity in larvae of the tenebrionid beetle, Z. atratus (Miller et al., 1996), and is considerably lower than the hundred or more nodules/individual seen in tobacco hornworms (Miller et al., 1994). In identical experiments with silkworms, we recorded about 80 nodules/individual (Stanley-Samuelson et al., 1997). It is clear that insect species differ in their expression of nodulation reactions to similar bacterial challenges. Turning to another gender difference in cicadas, we also note that males yielded about 100-fold more digestive PLA2 activity than females. PLA2 activity in the male cicada preparations (about 22 pmol/min/mg protein) was 2 to 3 times lower than the activity we recorded in similar preparations from two other species, tobacco hornworms, M. sexta (Rana et al., 1998) and mosquito larvae, Aedes aegypti (Nor Aliza and Stanley, 1998). The higher levels of digestive PLA2 activity in male cicadas, relative to females, may be indicative of more active feeding by males to support their energeti-
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cally expensive calling behavior. Recording low levels of PLA2 activity in female cicadas is consistent with our findings with tobacco hornworms and mosquito larvae, in which fasting individuals yielded much reduced PLA2 activity. The significance of assessing the presence of a digestive PLA2 in adult cicadas is indirectly connected to the biological roles of eicosanoids in these insects. As we have discussed for other species, the digestive PLA2 is an essential element of digestive physiology, responsible for hydrolyzing polyunsaturated fatty acids from dietary phospholipids to facilitate uptake of the free fatty acids into the cicada body. The presence of substantial digestive PLA2 activity in midguts of male cicadas indicates that dietary polyunsaturated fatty acids are important in the nutrition or energetics of these males. These dietary components are presumably important in eicosanoid biosynthesis, as well as several other aspects of physiology (Dadd, 1985). The dexamethasone dose–response curve and the results of the arachidonic acid rescue experiments strongly support the eicosanoid hypothesis. Moreover, a variety of eicosanoid biosynthesis inhibitors, specific for cyclooxygenase and lipoxygenase pathways, effectively retarded nodulation in adult cicadas. This apparent involvement of both cyclooxygenase and lipoxygenase products is probably due to the complexity of nodule formation, which involves many separate cellular actions, the inhibition of which may impact the overall nodulation process. Recognition of the biological significance of eicosanoids in insects and other invertebrates is rapidly increasing (Stanley-Samuelson, 1994a,b; Stanley-Samuelson and Pedibhotla, 1996; Stanley and Howard, 1998). Nonetheless, we have argued that the biochemistry of eicosanoids in insects is understudied (Miller et al., 1998). The presence of eicosanoid-precursor polyunsaturated fatty acids and eicosanoid biosynthesizing enzymes in insect tissues is a crucial point in discussions of the roles of eicosanoids in any aspect of insect physiology. In this work on cicadas, we used standard protocols to record the presence of arachidonic acid in cicada tissue phospholipids. As we observed with the desert cicada, Tibican dealbatus, the arachidonic acid was present in very low proportions of tissue phospholipids (Stanley-Samuelson et al., 1990). We also demonstrated that fat body preparations from M. septendecim are competent to biosynthesize eicosanoids from exogenous radioactive arachidonic acid. There is considerable variation in rates of prostaglandin biosynthesis among invertebrates. Total prostaglandin biosynthesis in the fat body preparations was on par with our findings with the desert cicada, T. dealbatus (Stanley-Samuelson et al., 1990), with fat body preparations from the silkmoth, B. mori, and with whole animal preparations of the lone star tick (Pedibhotla et al., 1995). On the other hand, the rates we observed with M. septendecim were lower than pro-
staglandin biosynthesis rates in tobacco hornworm hemocytes by approximately an order of magnitude (Gadelhak et al., 1995). Fat body is an immunity-conferring tissue in insects, and at least in the silkmoth fat body prostaglandins are thought to mediate induction of two genes for antibacterial proteins (Morishima et al., 1997). We note this work with cicada fat body does not directly demonstrate that cicada hemocytes are competent to produce eicosanoids. Nevertheless, these results indicate a tissue capacity for eicosanoid biosynthesis in cicadas, and we take these findings to support the eicosanoid hypothesis.
Acknowledgements We thank Dr Ralph Howard and Dr E.A. Heinrichs for reading and providing useful comments on a draft of this paper. This is paper no. 12,335 of the Nebraska Agricultural Research Division, and contribution no. 1,000 of the Department of Entomology. This work was supported by the Agricultural Research Division, UNL (Project NEB-17-054) and by USDA-ARS Specific Cooperative Agreement # 58-5430-5-115.
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