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Eicosanoid biosynthesis is activated via Toll, but not Imd signal pathway in response to fungal infection
Journal of Invertebrate Pathology 110 (2012) 382–388
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Eicosanoid biosynthesis is activated via Toll, but not Imd signal pathway in response to fungal infection Jung-A Park, Yonggyun Kim ⇑ Department of Bioresource Sciences, Andong National University, Andong 760-749, Republic of Korea
a r t i c l e
i n f o
Article history: Received 30 January 2012 Accepted 28 April 2012 Available online 5 May 2012 Keywords: Phospholipase A2 Eicosanoid Immune Spodoptera exigua Beauveria bassiana
a b s t r a c t Phospholipase A2 (PLA2) 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. PLA2 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 PLA2s 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 PLA2 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. Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction Insect immunity is exclusively innate and consists of cellular and humoral responses (Beckage, 2008). Upon microbial challenge after penetration into integument barrier, pattern recognition receptors specifically recognize each specific nonself type depending on surface molecular determinants varied among microbes (Medzhitov and Janeway, 1997, 2000). The recognition elicits its specific immune signal pathways, in which Toll and Imd pathways are crucial to activate immune responses especially in production of antimicrobial peptides (AMPs) in Drosophila and other insects (Shin et al., 2005; Lemaitre and Hoffmann, 2007; Zou et al., 2007; Tanaka et al., 2008). Toll signal pathway is primarily activated by Gram-positive bacterial and fungal infection, but Imd signal pathway is activated by Gram-negative bacteria in Drosophila (Lemaitre and Hoffmann, 2007). Eicosanoids consist of various oxygenated polyunsaturated fatty acids mainly derived from arachidonic acid (AA) (Stanley, 2005). Even though eicosanoids mediate various physiological processes in insects, they play crucial roles in immune responses (Stanley, 2011). Eicosanoids mediate various cellular immune responses. Miller et al. (1994) showed that eicosanoids mediate microaggregation and nodule formation to bacterial infection. In addition, ⇑ Corresponding author. Fax: +82 54 820 6320. E-mail address: [email protected] (Y. Kim). 0022-2011/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jip.2012.04.015
eicosanoids were reported to activate prophenoloxidase (PPO) and induce phagocytosis and cell spreading (Mandato et al., 1997). Hemocytic encapsulation of Drosophila melanogaster against parasitoid eggs was also known to be mediated by eicosanoids (Carton et al., 2002). A detailed molecular action of eicosanoids was analyzed in Spodoptera exigua, in which eicosanoids (especially, prostaglandins) induce cell lysis of oenocytoids that store PPO to be activated in the plasma by a series of catalytic activities of serine proteinases (Shrestha and Kim, 2008, 2009a). Eicosanoids also mediate humoral immune response in insects. Morishima et al. (1997) showed that eicosanoid biosynthesis inhibitors inhibit expression of antimicrobial peptides (AMPs), such as lysozyme and cecropin in Bombyx mori. Yajima et al. (2003) showed that Imd signal pathway is inhibited by eicosanoid biosynthesis inhibitor. In S. exigua, specific prostanglandins and leukotriene have been shown to mediate induction of AMP gene expressions (Shrestha and Kim, 2009b). In Tribolium castaneum, both Toll and Imd signal pathways respectively inducing specific AMP expressions activate eicosanoid biosynthesis in response to various bacterial infections (Shrestha and Kim, 2010). Phospholipase A2 (PLA2) catalyzes the first step of eicosanoid biosynthesis by hydrolyzing AA from membrane phospholipid at sn-2 position. Non-venom PLA2s have been identified in the genome of T. castaneum and known to be associated with immune responses (Shrestha et al., 2010). Tunaz et al. (2003) demonstrated that bacterial challenge significantly induces PLA2 activity in
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hemocytes of Manduca sexta. The significance of PLA2 activity against microbial challenge is further supported by pathogenic mechanism of an entomopathogenic bacterium, Xenorhabdus nematophila, in which the bacteria induce significant immunosuppression of S. exigua, but the addition of AA to the bacterial challenge reverses the pathogenic process (Park and Kim, 2000, 2003). PLA2 activity is required for defending entomopathogenic fungi because its specific inhibitor significantly enhances the fungal pathogenicity (Dean et al., 2002; Lord et al., 2002; Tunaz, 2006). Though we know much about immune signal pathways and eicosanoid effects in insect system, it is still little understood on their interactions. Here, we demonstrate a functional link between immune signal pathway and PLA2 activation in response to fungal challenge in S. exigua. To link these immune events, we tested effect of RNA interference (RNAi) of each immune signal pathway on modulating PLA2 activity. This would be the first report on a functional relationship between Toll signal pathway and activation of PLA2 in response to fungal challenge in insects.
Table 1 Nucleotide sequences of PCR primers used in this study. Genes
Primer sequences
SeToll
50 -GAG TGC GAC TGT ACA ATG G-30 50 -GGT CGC ATC CAT CGG TAT TC-30 50 -TGT GAT CTA GCA AGT GCA TTG -30 50 -ACT TCA ATT CCG TCT TCT GTC-30 50 -ATG GTC GCC AAG TTG TTC GTG-30 50 -CTC CTG CGC GGT GTT CTG CA-30 50 -GTC CCT CTC TGT CCT GAA GG-30 50 -CAG AAA CAC GAA GAA AGA TGG-30 50 -GAT GTT CTG GCG CAG CTG TC-30 50 -CCG GCT GAA CGC AAA CAC AG-30 50 -GCT TTC CTC TCC AGG AAT ATG-30 50 -CCT TAG AGT AAA TCC AGT GG-30 50 -TCC CGA ATG TGC CCA ACT TC-30 50 -GAA AGA TCT GCC GAA AGT AAG-30 50 -CGT GGA CAT CTT CAG GGC C-30 50 -GTC GTG TTC AAT GCC ACC G-30 50 -AAG ACC AGG GCG AGT ACA AG-30 50 -AGC GAC ATG AAC CAA GGT TTC-30 50 -ATC GTT TAG CTT CGT GTT CGC-30 50 -CTT TCT TTT ACC ACA CGG TTG-30 50 -TCA GTC ATG AAG GCT TGC GTA-30 50 -TCG CAC ACA TTG GCA TCC ATT G-30
SeRelish SeApolipophorin III SeTransferrin-1 SeTransferrin-2 SeAttacin-A SeAttacin-B SeGloverin SeHemolin SeCecropin
2. Materials and methods
SeGallerimycin
2.1. Insect and fungus cultures The beet armyworm, S. exigua, used in this study was originated from a field population infesting welsh onion (Allium fistulosum) in Andong, Korea. The larvae were reared on an artificial diet (Goh et al., 1990) at 25 °C and the adults were fed 10% sucrose. An entomopathogenic fungus, Beauveria bassiana, was cultured on solid potato dextrose agar (PDA) medium (5% potato extract, 0.5% dextrose, 1.7% agar) at 25 °C for 7 days. The cultured fungal colonies were resuspended with phosphate buffered saline (PBS, 50 mM phosphate, 0.7% NaCl, pH 7.0). Mixture of conidia and mycelia was filtered using a filter paper (pore size = 20 lm) and the resulting flow-through suspension was used for further study. 2.2. Enzyme activity measurement PLA2 activity was measured by spectrofluorometry using a pyrene-labeled phospholipid [1-hexadecanoyl-2-(1-pyrenedecanoyl)sn-glycerol-3-phosphatidyl choline] as a substrate in the presence of bovine serum albumin (Radvanyi et al., 1989). The fluorometric phospholipid was dissolved in ethanol to prepare 10 mM stock solution and 10% of bovine serum albumin (BSA) was prepared in sterilized distilled water. The reaction mixture (2 ml) was prepared in a cuvette by sequentially adding 1946 ll of 50 mM Tris–HCl buffer (pH 7.0), 20 ll of 10% BSA, 12 ll of 1 M CaCl2, and 20 ll of enzyme extract. The reaction was initiated by addition 2 ll of 10 mM pyrene substrate and subsequently fluorescence intensity was monitored with an Aminco Bowmen Series 2 luminescence spectrometer (FA257, Spectronic Instruments, USA) using excitation and emission wavelengths of 345 and 398 nm, respectively. The specific enzyme activity was calculated in pmol/min according to method of Radvanyi et al. (1989). For pH preference, different pH solutions were prepared with stock solutions of sodium phosphate buffer using Henderson–Hasselbalch equation. To check inhibition of its activity, 2 ll of test chemical was used in above described reaction mixture. 2.3. RT-PCR of different antimicrobial peptides (AMPs) Total RNA was isolated from larvae of S. exigua with Trizol reagent (Invitrogen, Carlsbad, CA, USA). First strand cDNA was synthesized with 1 ll of the extracted total RNA by using RT-Premix oligo-dT (50 -CCA GTG AGC AGA GTG ACG AGG ACT CGA GCT CAA GC TTT TTT TTT TTT TTT T-30 , Intron, Seoul, Korea). AMP genes were
amplified with gene-specific primers (Table 1). PCR was performed with 35 cycles of denaturation (94 °C, 1 min), annealing (50 °C, 1 min), and extension (72 °C, 1 min). As a control, expression of b-actin was analyzed with gene-specific primers (50 -TGG CAC CAC ACC TTC TAC TAC-30 and 50 -CAT GAT CTG GGT CAT CTT CT-30 ) to confirm the integrity of cDNA. 2.4. RNA interference (RNAi) Double stranded RNAs (dsRNAs) of SeToll, SeRelish and a viral gene ORF302 as a control dsRNA (Park and Kim, 2010) were prepared according to the manufacturer’s instruction of Megascript RNAi kit (Ambion, Massachusetts, TX, USA). Briefly, each gene fragment (342 bp for SeToll, 446 bp for SeRelish and 419 bp for ORF302) produced by PCR using each pair of the gene-specific primers was cloned into pCR2.1 vector (Invitrogen, Carlsbad, CA, USA). To screen the insert direction, plasmid DNAs containing recombinant SeToll, SeRelish and ORF302 were analyzed by restriction enzyme digestion using Sal I, Bam HI (Bioneer, Daejon, Korea) and Dde I (BEAMS Bio, Seoul, Korea), respectively. Sense and antisense strands were synthesized using T7 RNA polymerase at 37 °C for 4 h. To be effective, these dsRNAs were injected twice to S. exigua larvae. The first was at the 3 days old 4th instar (last day of this instar) and the second shot was at one day old 5th instar. dsRNA was prepared with Metafectene PRO (Biontex, Plannegg, Germany) in 1:1 volume ratio and incubated at 25 ± 1 °C for 20 min. Two l of the dsRNA (50 ng/ll) solution was injected into larval hemocoel at every time. Knockdown of specific mRNA was evaluated by RT-PCR every 12 or 24 h after the second injection of the dsRNA. 2.5. Nodule formation assay Nodulation assay was performed by injecting fungal suspension in a volume of 5 ll (5 104 conidia/larva) through an abdominal proleg using a 10 ll Hamilton syringe (Hamilton, Reno, Nevada, USA). After 8 h incubation at 25 °C, the treated larvae were dissected on dorsal side and the melanized nodules on its gut and fat body were initially counted under a stereoscopic microscope (SZX9, Olympus, Tokyo, Japan) at 50x magnification. After the alimentary canal was removed, nodules in the previously unexposed areas and remaining internal tissues were then counted and added to the initial count. Each treatment consisted of 10 test larvae.
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2.6. Data analysis Treatment means and variances were analyzed in one-way ANOVA by PROC GLM of SAS program (SAS Institute, 1989). All means were compared by least squared difference (LSD) tests at Type I error = 0.05. 3. Results 3.1. PLA2 activities of hemocyte and fat body are enhanced by a fungal infection Hemocyte and fat body are immune-associated tissues and analyzed in their PLA2 activity in S. exigua (Fig. 1). With increase of substrate concentration, specific PLA2 activities of these two tissues and plasma increased and reached their respective maximal levels, in which maximal enzyme activity of hemocyte PLA2 was greater than those of plasma and fat body (Fig. 1B). Based on these substrate-enzyme kinetic curves, Michaelis–Menten parameters of different PLA2s were estimated. Hemocyte PLA2 was significantly
Table 2 Enzyme kinetic parameters of PLA2s extracted from different tissues of fifth instar Spodoptera exigua. PLA2 source
Enzyme kinetic parametersA KM (lM)
Hemocyte Plasma Fat body
Vm (pmol/min/lg) a
0.34 ± 0.11 0.25 ± 0.05b 0.26 ± 0.09b
a
3.11 ± 0.23 2.06 ± 0.07b 1.79 ± 0.12b
I50 (nM) against BZAB 16.52 ± 3.71a 1.26 ± 3.19b 1.62 ± 6.81b
A Different letters followed by standard deviations are significantly different in means in each parameter at Type I error = 0.05 (LSD test). B Benzylideneacetone.
different from those of fat body and plasma in substrate affinity (KM) and maximal enzyme catalytic capacity (Vm) (Table 2). To determine the difference in PLA2s of different tissues, their susceptibilities to a specific PLA2 inhibitor were analyzed. Benzylideneacetone (BZA) is a specific PLA2 inhibitor derived from an entomopathogenic bacterium, X. nematophila, culture broth (Ji et al. 2004). It significantly inhibited PLA2 activities of all three tissue sources (Fig. 1B). Fat body and plasma PLA2s were more susceptible than that of hemocyte (Table 2). PLA2 activity increased in the immune-associated tissues, fat body and hemocyte, and in plasma after injection of B. bassiana (Fig. 2). The induction of PLA2 activity occurred as early as 2–4 h after the fungal infection. 3.2. RNAi of Toll signal suppresses specific AMP gene expressions and PLA2 activation
Fig. 1. PLA2 activities of the immune-associated tissues of fat body (FB) and hemolymph separated into hemocyte (HC) and plasma (PL) of fifth instar larvae of Spodoptera exigua. (A) Change in specific enzyme activities with increase of substrate concentration in different tissue type PLA2s. (B) Inhibition of PLA2 activities of different tissue type PLA2 by a specific PLA2 inhibitor, benzylideneacetone (BZA). Each measurement consisted of three independent replications. Different letters above standard deviation bars indicate significant difference among means at Type I error = 0.05 (LSD test).
To link immune signal pathways to PLA2 activation, RNAi was conducted against Toll and Imd pathways, respectively (Fig. 3). Both SeToll and SeRelish (a component of Imd pathway) were markedly expressed in response to bacterial immune challenges (Hwang, 2011). However, challenge with B. bassiana strongly induced expression of SeToll gene, but weakly induced expression of SeRelish. The expression of SeToll was specifically inhibited by dsRNA specific to SeToll, but not by dsRNA specific to SeRelish (Fig. 3A). Under theses RNAi conditions, we analyzed expressions of nine AMP genes in fat body and hemocyte (Fig. 3B). In both tissues, both attacins and cecropin were highly inducible to fungal infection, whereas gallerimycin, hemolin, and transferrin I were slightly inducible. However, apolipophorin III, gloverin, and transferrin II were strongly expressed irrespective of the fungal infection. When dsRNA specific to SeRelish or SeToll were respectively treated, these AMPs showed different suppression patterns. Apolipophorin III and transferrin II were insensitive to either dsRNAs due to their strong constitutive expressions. The other seven AMPs were inhibited in their expression by dsRNA specific to SeToll in both tissues. By contrast, dsRNA specific to SeRelish inhibited expression of only cecropin in hemocytes. Such inhibitory patterns were more evident in hemocytes than fat body. These results confirmed independent immune signaling pathways of Toll and Imd in S. exigua. Under these specific RNAi conditions, PLA2 activation of different tissues in response to fungal infection was analyzed (Fig. 4). dsRNA specific to SeToll significantly inhibited the activation of PLA2s of all tested tissues in response to the fungal infection. However, dsRNA specific to SeRelish did not suppress PLA2 activation in tested tissues. 3.3. Hemocyte nodulation is inhibited by dsRNA treatment specific to Toll pathway, but not to Imd pathway Eicosanoids mediate hemocyte nodule formation in response to bacterial or fungal infection (see Introduction). Inhibition of PLA2
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4. Discussion
Fig. 2. Induction of PLA2 activity in different PLA2s extracted from hemocyte (HC), plasma (PL), and fat body (FB). An entomopathogenic fungus, Beauveria bassiana, was hemocoelically injected into fifth instar larvae of Spodoptera exigua in a dose of 1.1 105 conidia and incubated for different periods at 25 °C. Each measurement consisted of three independent replications. Different letters above standard deviation bars indicate significant difference among means at Type I error = 0.05 (LSD test).
activation prevents eicosanoid biosynthesis, which would be expected to result in suppression of nodule formation. In response to fungal infection, S. exigua larvae significantly induced nodule formation compared to solvent injection (Fig. 5A). The nodule formation was significantly suppressed in the larvae treated with dsRNA specific to SeToll. By contract, dsRNA specific to SeRelish did not suppress the nodule formation. Exogenous AA addition significantly rescued nodule formation inhibited by RNAi of SeToll gene expression (Fig. 5B).
Eicosanoids mediate both cellular and humoral immune responses against various microbes (Stanley, 2005). Biosynthesis of eicosanoids is inducible to pathogen infection presumably after nonself recognition (Tunaz et al., 2003; Park and Kim, 2011). Depending on surface molecular structures, various microbes are discriminated by specific pattern recognition receptors (Medzhitov and Janeway, 2000). After recognition, two main immune signaling pathways, Toll and Imd pathways, lead to expression of various AMP genes depending on microbes (Lemaitre and Hoffmann, 2007). Thus, eicosanoid biosynthesis and Toll/Imd pathways may be associated after nonself recognition. This functional link was demonstrated in T. castaneum in response to various bacterial challenge, in which both Toll and Imd pathways are associated with PLA2 activation (Shrestha and Kim, 2010). This current study proved that only Toll signal pathway activated eicosanoid biosynthesis in response to a fungal infection in S. exigua. First, two main immune effectors (hemocyte and fat body) that possessed signal components of Toll/Imd pathways showed their specific PLA2 activities that were significantly induced by fungal infection. The activated PLA2 would catalyze AA release from phospholipids for subsequent eicosanoid biosynthesis. Second, the fungal infection induced expression of several AMPs that were mostly modulated by expression of Toll. The AMPs induced by Toll expression were distinct from those induced by Imd component (SeRelish). Third, RNAi experiments showed that dsRNAToll significantly inhibited activation of PLA2 in response to the fungal infection, but dsRNARelish did not. Fourth, hemocyte nodule formation induced by the fungal infection was significantly inhibited by dsRNAToll, but not by dsRNARelish. Lastly, the suppressed nodule formation by dsRNAToll was rescued by addition of AA. PLA2s constitute a superfamily of enzymes that catalyze phospholipid hydrolysis at the sn-2 ester bond and have been classified into at least 15 groups comprising of four main types: secretory (sPLA2), cytosolic Ca2+-dependent (cPLA2), cytosolic Ca2+-independent (iPLA2), and PAF acetyl hydrolase/oxidized lipid (LpPLA2) PLA2 (Burke and Dennis, 2009). Immune-associated PLA2s were detected from hemocyte, plasma, and fat body in S. exigua. These PLA2 activities were significantly enhanced by infection of B. bassiana. However, these different tissue PLA2s showed different enzyme kinetics, in which hemocyte PLA2 showed lower substrate affinity, higher catalytic capacity, and higher sensitivity to BZA (a specific PLA2 inhibitor) than plasma and fat body PLA2s. BZA is a competitive inhibitor against catalytic activity of PLA2 associated with insect immune responses (Shrestha and Kim, 2007; Kwon and Kim, 2008). Plasma and fat body PLA2s were not different with each other in the enzyme properties, such as KM, Vm, and inhibitor susceptibility. Recently four immune-associated PLA2s are identified in T. castaneum (Shrestha et al., 2010). All these PLA2s are secretory type and susceptible to BZA (Shrestha and Kim, 2010). Antibody raised from a T. castaneum sPLA2 did not reacted with PLA2 extracts of S. exigua (data not shown). This suggests that immune-associated PLA2s of S. exigua may have unique molecular forms. Also, the difference between hemocyte and other tissue PLA2s suggests that more than one type of PLA2s may play crucial role in immune responses in S. exigua. RNAi experiments suggest that Toll and Imd signal pathways act in immune responses of S. exigua. Toll and Imd immune signal pathways are well known in Drosophila (Lemaitre et al., 1995; Michel et al., 2001; Tanji et al., 2007). Upon infection of Gram-positive bacteria (Lys-type peptidoglycan) or fungi, Toll pathway is activated by prior proteolytic activation of proSpätzle in the plasma and its subsequent binding to the Toll receptor leads to an intracellular signaling cascade, which results in degradation of Cactus (an
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Fig. 3. RNA interference of immune signal genes (SeRelish and SeToll) suppresses gene expressions of different antimicrobial peptides (AMPs) in the fifth instar Spodoptera exigua in response to infection of Beauveria bassiana (Bb). Nine AMPs of S. exigua in this study included Apolipophorin III (SeApoLp III), attacin A (SeAtt A), attacin B (SeAtt B), cecropin (SeCec), gloverin (SeGlv), gallerimycin (SeGal), hemolin (SeHem), transferrin I (SeTf I), and transferrin II (SeTf II). b-Actin was used in this RT-PCR to confirm the integrity of cDNA preparation. (A) Specific interference of SeRelish or SeToll expressions by their specific double stranded RNAs (dsRNAs). A viral gene (ORF302) was used for dsRNA control. RT-PCRs were performed at 24 h after the dsRNA injections (200 ng per larva). (B) Inhibition of AMP gene expression by treatment of dsRNA specific to SeRelish or SeToll. At 24 h after dsRNA injection, larvae were individually injected with Bb in 5 104 conidia and incubated for 8 h at 25 °C. The treated larvae were then analyzed for AMP expressions by RT-PCR. Control represents a sham injection using PBS.
inhibitor of kB) and allows NF-jB (Dif or Dorsal) to migrate into nucleus to induce AMP gene expressions (Hoffmann, 2003). By contrast, Imd pathway is activated by the challenge of Gram-negative bacteria (diaminopimelate-type peptidoglycan), in which cooperative activity of PGRP-LC and PGRP-LE recognition receptors
activates unidentified transmembrane receptor and subsequent an adaptor protein, Imd, associates with FADD with its death domain (Leclerc and Reichhart, 2004). Thereafter a sequential activation of TAK1, Dredd, and IKK complex cleaves inactive NF-jB (Relish), which migrates into the nucleus to induce specific AMPs (Tanji
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Fig. 5. Hemocyte nodule formation in response to Beauveria bassiana (Bb) infection is linked to Toll signal pathway in Spodoptera exigua. At 24 h after dsRNA injection, larvae were individually injected with Bb in 5 104 conidia and incubated for 8 h at 25 °C. Each nodulation assay consisted of three independent replications. Different letters above standard deviation bars indicate significant difference among means at Type I error = 0.05 (LSD test). (A) Specific inhibition by treatment of dsRNA specific to SeToll. (B) Rescue effect on the suppressed nodulation by the addition of arachidonic acid (100 ng per larva). Control represents Bb injection.
Fig. 4. Induction of PLA2 activities of hemocyte (HC), plasma (PL), and fat body (FB) via Toll signal pathway in response to Beauveria bassiana (Bb) infection in fifth instar Spodoptera exigua. At 24 h after dsRNA injection, larvae were individually injected with Bb in 5 104 conidia and incubated for 8 h at 25 °C. Each enzyme activity measurement consisted of three independent replications. Different letters above standard deviation bars indicate significant difference among means at Type I error = 0.05 (LSD test).
and Ip, 2005). In response to B. bassiana, S. exigua larvae expressed all nine tested AMPs, in which except apolipophorin III and transferrin II that were constitutively expressed from a semi-RT-PCR analysis, seven AMPs were controlled under expression of SeToll expression because expressions of these seven AMPs were inhibited by dsRNAToll. However, the contribution of Imd pathway in the fungal infection was not much significant because itsnockdown by dsRNARelish inhibited expression of cecropin, but the AMP expression was also controlled by Toll pathway. These results suggest that Toll and Imd signal pathways independently mediate immune signals in S. exigua, in which Toll signal pathway mediates immune signal in response to infection of B. bassiana. Though PLA2s of three tissues were activated by infection of B. bassiana, dsRNAToll significantly inhibited the enzyme induction. By contrast, dsRNARelish did not inhibit the induction of PLA2 activity.
In addition, dsRNAToll significantly inhibited hemocyte nodule formation against the fungal infection, which was rescued by addition of AA. This suggests that downstream signal of Toll pathway may activate the immune-associated PLA2, which in turn activate biosynthesis of eicosanoids that mediate the cellular immune. Though little is known in cross-talk between Toll signal and PLA2 activation in invertebrates, mammalian immune cells show that PLA2 is activated by its phosphorylation due to kinases activated by Myd88 that is an adaptor molecule in a downstream of Toll-like receptor in response to immune challenge (Qi and Shelhamer, 2005). In T. castaneum, PLA2 was activated by de novo synthesis of PLA2 or by translocation to cell membrane (Shrestha and Kim, 2010), in which Toll signal may modulate both activations. These facts in other systems suggest that Myd88 may be an adaptor between Toll signal and PLA2 activation in S. exigua and that knockdown of Myd88 expression may uncouple the PLA2 activation by Toll signal pathway. Furthermore, in mammalian immune cells, Myd88 activates MAPKs, which subsequently activates cPLA2 by phosphorylation (Clark et al., 1991; Kramer et al., 1996). This suggests that there may be kinase(s) that is activated by Toll pathway and finally activates PLA2 of S. exigua. Our data provide the first insight of Toll immune signaling in its role other than AMP production in insects in response to fungal infection. Also, the specific coupling of Toll signaling and PLA2
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activation in fungal infection makes clear the immune signal pathway from recognition to eicosanoid biosynthesis because little information is available to understand how pathogen recognition activates eicosanoid biosynthesis even though there are quite many studies on immunological roles of eicosanoids in insects (Stanley, 2011). To better understand eicosanoid function in insect immune responses, further studies should be focused on how Toll or Imd signaling pathways activate catalytic activity of PLA2 by identifying adaptor molecules that play a role in conveying recognition signal to eicosanoid biosynthesis pathway. Acknowledgments This study was funded by a Basic Research Grant of National Research Foundation, Korea to Y. Kim. J. Park was supported by the second stage BK21 program of the Ministry of Education, Science and Technology, Korea. References Beckage, N.E., 2008. Insect Immunology. Academic press, New York. Burke, J.E., Dennis, E.A., 2009. Phospholipase A2 structure/function, mechanism, and signaling. J. Lipid Res. 50, S237–S242. Carton, Y., Frey, F., Stanley, D.W., Vass, E., Nappi, A.J., 2002. Dexamethasone inhibition of the cellular immune response of Drosophila melanogaster against a parasitoid. J. Parasitol. 88, 405–407. Clark, J.D., Lin, L.L., Kriz, R.W., Ramesha, C.S., Sultzman, L.A., Lin, A.Y., Minola, N., Knopf, J.L., 1991. A novel arachidonic acid-selective cytosolic PLA2 contains a Ca2+-dependent translocation domain with homology to PKC and GAP. Cell 65, 1043–1051. Dean, P., Gadsden, J.C., Richards, E.H., Edwards, J.P., Charnley, A.K., Reynolds, S.E., 2002. Modulation by eicosanoid biosynthesis inhibitors of immune responses by the insect Manduca sexta to the pathogenic fungus Metarhizium anisopliae. J. Invertebr. Pathol. 79, 93–101. Goh, H.G., Lee, S.G., Lee, B.P., Choi, K.M., Kim, J.H., 1990. Simple mass-rearing of beet armyworm, Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae), on an artificial diet. Kor. J. Appl. Entomol. 29, 180–183. Hoffman, J.A., 2003. The immune response of Drosophila. Nature 426, 33–38. Hwang, J., 2011. Transcriptional control of humoral immune responses in the beet armyworm, Spodoptera exigua. Master Thesis. Andong National University, Andong, Republic of Korea. Ji, D., Yi, Y., Kim, G.H., Choi, Y.H., Kim, P., Baek, N.I., Kim, Y., 2004. Identification of an antibacterial compound, benzylideneacetone, from Xenorhabdus nematophila against major plant-pathogenic bacteria. FEMS Microbiol. Lett. 239, 241–248. Kramer, R.M., Roberts, E.F., Um, S.L., Borsch-Houbold, A.G., Watson, S.P., Fisher, M.J., Jakubowski, J.A., 1996. P38 mitogen-activated protein kinase phosphorylates cytosolic phospholipase A2 (cPLA2) in thrombin-stimulated platelets. Evidence that proline-directed phosphorylation is not required for mobilization of arachidonic acid by cPLA2. J. Biol. Chem. 271, 27723–27729. Kwon, B., Kim, Y., 2008. Benzylideneacetone, an immunosuppressant, enhances virulence of Bacillus thuringiensis against beet armyworm (Lepidoptera: Noctuidae). J. Econ. Entomol. 101, 36–41. Leclerc, V., Reichhart, J.M., 2004. The immune response of Drosophila melanogaster. Immunol. Rev. 198, 58–71. Lemaitre, B., Hoffmann, J., 2007. The host defense of Drosophila melanogaster. Annu. Rev. Immunol. 25, 697–743. Lemaitre, B., Meister, M., Govind, S., Georgel, P., Steward, R., Reichhart, J.M., Hoffmann, J.A., 1995. Functional analysis and regulation of nuclear import of dorsal during the immune response in Drosophila. EMBO J. 14, 536–545. Lord, J.C., Anderson, S., Stanley, D.W., 2002. Eicosanoids mediate Manduca sexta cellular response to the fungal pathogen Beauveria bassiana: a role for lipoxygenase pathway. Arch. Insect Biochem. Physiol. 51, 46–54. Mandato, G.A., Diehl-Jones, L., Moore, S.J., Downer, R.G., 1997. The effect of eicosanoid biosynthesis inhibitors on prophenoloxidase activation, phagocytosis and cell spreading in Galleria mellonella. J. Insect Physiol. 43, 1–8. Medzhitov, R., Janeway Jr., C.A., 1997. Innate immunity: the virtues of a nonclonal system of recognition. Cell 91, 295–298. Medzhitov, R., Janeway Jr., C.A., 2000. Innate immunity recognition: mechanisms and pathways. Immunol. Rev. 173, 89–97. Michel, T., Reichhart, J.M., Hoffman, J.A., Royet, J., 2001. Drosophila Toll is activated by gram-positive bacteria through a circulating peptidoglycan recognition protein. Nature 414, 756–759.
Miller, J.S., Nguyen, T., Stanley-Samuelson, D.W., 1994. Eicosanoids mediate insect nodulation responses to bacterial infections. Proc. Natl. Acad. Sci. USA 91, 12418–12422. Morishima, I., Yamano, Y., Inoue, K., Matsuo, N., 1997. Eicosanoids mediate induction of immune genes in the fat body of the silkworm, Bombyx mori. FEBS Lett. 419, 83–86. Park, B., Kim, Y., 2010. Transient transcription of a putative RNase containing BEN domain encoded in Cotesia plutellae bracovirus induces an immunosuppression of the diamondback moth, Plutella xylostella. J. Invertebr. Pathol. 105, 156–163. Park, J., Kim, Y., 2011. Benzylideneacetone suppresses both cellular and humoral immune responses of Spodoptera exigua and enhances fungal pathogenicity. J. Asia Pac. Entomol. 14, 423–427. Park, Y., Kim, Y., 2000. Eicosanoids rescue Spodoptera exigua infected with Xenorhabdus nematophilus, the symbiotic bacteria to the entomopathogenic nematode, Steinernema carpocapsae. J. Insect Physiol. 46, 1469–1476. Park, Y., Kim, Y., 2003. Xenorhabdus nematophila inhibits p-bromophenacyl bromide (BPB)-sensitive PLA2 of Spodoptera exigua. Arch. Insect Biochem. Physiol. 54, 134–142. Qi, H.Y., Shelhamer, J.H., 2005. Toll-like receptor 4 signaling regulates cytosolic phospholipase A2 activation and lipid generation in lipopolysaccharidestimulated macrophages. J. Biol. Chem. 280, 38969–38975. Radvanyi, F., Jordan, L., Russo-Marie, F., Bon, C., 1989. A sensitive and continuous fluorometric assay for phospholipase A2 using pyrene-labeled phospholipids in the presence of serum albumin. Anal. Biochem. 177, 103–109. SAS Institute, Inc. 1989. SAS/STAT User’s Guide, release 6.03 ed. SAS Institute, Cary, NC. Shin, S.W., Kokoza, V., Bian, G., Cheon, H.M., Kim, Y.J., Raikhel, A., 2005. REL1, a homologue of Drosophila Dorsal, regulates Toll antifungal immune pathway in the female mosquito, Aedes aegypti. J. Biol. Chem. 280, 16499–16507. Shrestha, S., Kim, Y., 2007. An entomopathogenic bacterium, Xenorhabdus nematophila, inhibits hemocyte phagocytosis of Spodoptera exigua by inhibiting phospholipase A2. J. Invertebr. Pathol. 96, 64–70. Shrestha, S., Kim, Y., 2008. Eicosanoid mediates prophenoloxidase release from oenocytoids in the beet armyworm, Spodoptera exigua. Insect Biochem. Mol. Biol. 38, 99–112. Shrestha, S., Kim, Y., 2009a. Oenocytoid cell lysis to release prophenoloxidase in induced by eicosanoid via protein kinase C. J. Asia Pac. Entomol. 12, 301–305. Shrestha, S., Kim, Y., 2009b. Various eicosanoids modulate the cellular and humoral immune responses of the beet armyworm, Spodoptera exigua. Biochem. Biophys. Biotech. 73, 2077–2084. Shrestha, S., Kim, Y., 2010. Activation of immune-associated phospholipase A2 is functionally linked to Toll/Imd signal pathways in the red flour beetle, Tribolium castaneum. Dev. Comp. Immunol. 34, 530–537. Shrestha, S., Park, Y., Stanley, D., Kim, Y., 2010. Genes encoding phospholipase A2 mediate insect nodulation reactions to bacterial challenge. J. Insect Physiol. 56, 324–332. Stanley, D.W., 2005. Eicosanoids. In: Gilbert, L.I., Iatrou, K., Gill, S.S. (Eds.), Comprehensive Insect Molecular Science, vol. 4. Elsevier, Amsterdam, pp. 307–339. Stanley, D.W., 2011. Eicosanoids: progress towards manipulating insect immunity. J. Appl. Entomol. 135, 534–545. Tanaka, H., Ishibashi, J., Funjuta, K., Nakajima, Y., Sagisaka, A., Tonomoto, K., Suzuki, N., Yoshiyama, M., Kaneko, Y., Iwasaki, T., Sunagawa, T., Yamaji, K., Asaoka, A., Mita, K., Yamakawa, M., 2008. A genome-wide analysis of genes and gene families involved in innate immunity of Bombyx mori. Insect Biochem. Mol. Biol. 38, 1087–1110. Tanji, T., Hu, X., Weber, A.N., Ip, Y.T., 2007. Toll and IMD pathway synergistically activate an innate immune response in Drosophila melanogaster. Mol. Cell. Biol. 27, 4578–4588. Tanji, T., Ip, Y.T., 2005. Regulators of the Toll and Imd pathways in the Drosophila innate immune response. Trends Immunol. 26, 193–198. Tunaz, H., 2006. Eicosanoid biosynthesis inhibitors influence mortality of Pieris brassicae larvae co-injected with fungal conidia. Arch. Insect Biochem. Physiol. 63, 93–100. Tunaz, H., Park, Y., Buyukguzel, K., Bedick, J.C., Noraliza, A.R., 2003. Eicosanoids in insect immunity: bacterial infection stimulates hemocytic phospholipase A2 activity in tobacco hornworms. Arch. Insect Biochem. Physiol. 52, 1–6. Yajima, M., Takada, M., Takahashi, N., Kikuchi, H., Natori, S., Oshima, Y., Kurata, S., 2003. A newly established in vitro culture using transgenic Drosophila reveals functional coupling between the phospholipase A2-generated fatty acid cascade and lipopolysaccharide-dependent activation of the immune deficiency (Imd) pathway in insect immunity. Biochem. J. 371, 205-201. Zou, Z., Evans, J.D., Lu, Z., Zhao, P., Williams, M., Sumathipala, N., Hetru, C., Hultmark, D., Jiang, H., 2007. Comparative genomic analysis of the Tribolium immune system. Genomics Biol. 8, R177.
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Eicosanoid biosynthesis is activated via Toll, but not Imd signal pathway in response to fungal infection Jung-A Park, Yonggyun Kim ⇑ Department of Bioresource Sciences, Andong National University, Andong 760-749, Republic of Korea
a r t i c l e
i n f o
Article history: Received 30 January 2012 Accepted 28 April 2012 Available online 5 May 2012 Keywords: Phospholipase A2 Eicosanoid Immune Spodoptera exigua Beauveria bassiana
a b s t r a c t Phospholipase A2 (PLA2) 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. PLA2 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 PLA2s 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 PLA2 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. Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction Insect immunity is exclusively innate and consists of cellular and humoral responses (Beckage, 2008). Upon microbial challenge after penetration into integument barrier, pattern recognition receptors specifically recognize each specific nonself type depending on surface molecular determinants varied among microbes (Medzhitov and Janeway, 1997, 2000). The recognition elicits its specific immune signal pathways, in which Toll and Imd pathways are crucial to activate immune responses especially in production of antimicrobial peptides (AMPs) in Drosophila and other insects (Shin et al., 2005; Lemaitre and Hoffmann, 2007; Zou et al., 2007; Tanaka et al., 2008). Toll signal pathway is primarily activated by Gram-positive bacterial and fungal infection, but Imd signal pathway is activated by Gram-negative bacteria in Drosophila (Lemaitre and Hoffmann, 2007). Eicosanoids consist of various oxygenated polyunsaturated fatty acids mainly derived from arachidonic acid (AA) (Stanley, 2005). Even though eicosanoids mediate various physiological processes in insects, they play crucial roles in immune responses (Stanley, 2011). Eicosanoids mediate various cellular immune responses. Miller et al. (1994) showed that eicosanoids mediate microaggregation and nodule formation to bacterial infection. In addition, ⇑ Corresponding author. Fax: +82 54 820 6320. E-mail address: [email protected] (Y. Kim). 0022-2011/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jip.2012.04.015
eicosanoids were reported to activate prophenoloxidase (PPO) and induce phagocytosis and cell spreading (Mandato et al., 1997). Hemocytic encapsulation of Drosophila melanogaster against parasitoid eggs was also known to be mediated by eicosanoids (Carton et al., 2002). A detailed molecular action of eicosanoids was analyzed in Spodoptera exigua, in which eicosanoids (especially, prostaglandins) induce cell lysis of oenocytoids that store PPO to be activated in the plasma by a series of catalytic activities of serine proteinases (Shrestha and Kim, 2008, 2009a). Eicosanoids also mediate humoral immune response in insects. Morishima et al. (1997) showed that eicosanoid biosynthesis inhibitors inhibit expression of antimicrobial peptides (AMPs), such as lysozyme and cecropin in Bombyx mori. Yajima et al. (2003) showed that Imd signal pathway is inhibited by eicosanoid biosynthesis inhibitor. In S. exigua, specific prostanglandins and leukotriene have been shown to mediate induction of AMP gene expressions (Shrestha and Kim, 2009b). In Tribolium castaneum, both Toll and Imd signal pathways respectively inducing specific AMP expressions activate eicosanoid biosynthesis in response to various bacterial infections (Shrestha and Kim, 2010). Phospholipase A2 (PLA2) catalyzes the first step of eicosanoid biosynthesis by hydrolyzing AA from membrane phospholipid at sn-2 position. Non-venom PLA2s have been identified in the genome of T. castaneum and known to be associated with immune responses (Shrestha et al., 2010). Tunaz et al. (2003) demonstrated that bacterial challenge significantly induces PLA2 activity in
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hemocytes of Manduca sexta. The significance of PLA2 activity against microbial challenge is further supported by pathogenic mechanism of an entomopathogenic bacterium, Xenorhabdus nematophila, in which the bacteria induce significant immunosuppression of S. exigua, but the addition of AA to the bacterial challenge reverses the pathogenic process (Park and Kim, 2000, 2003). PLA2 activity is required for defending entomopathogenic fungi because its specific inhibitor significantly enhances the fungal pathogenicity (Dean et al., 2002; Lord et al., 2002; Tunaz, 2006). Though we know much about immune signal pathways and eicosanoid effects in insect system, it is still little understood on their interactions. Here, we demonstrate a functional link between immune signal pathway and PLA2 activation in response to fungal challenge in S. exigua. To link these immune events, we tested effect of RNA interference (RNAi) of each immune signal pathway on modulating PLA2 activity. This would be the first report on a functional relationship between Toll signal pathway and activation of PLA2 in response to fungal challenge in insects.
Table 1 Nucleotide sequences of PCR primers used in this study. Genes
Primer sequences
SeToll
50 -GAG TGC GAC TGT ACA ATG G-30 50 -GGT CGC ATC CAT CGG TAT TC-30 50 -TGT GAT CTA GCA AGT GCA TTG -30 50 -ACT TCA ATT CCG TCT TCT GTC-30 50 -ATG GTC GCC AAG TTG TTC GTG-30 50 -CTC CTG CGC GGT GTT CTG CA-30 50 -GTC CCT CTC TGT CCT GAA GG-30 50 -CAG AAA CAC GAA GAA AGA TGG-30 50 -GAT GTT CTG GCG CAG CTG TC-30 50 -CCG GCT GAA CGC AAA CAC AG-30 50 -GCT TTC CTC TCC AGG AAT ATG-30 50 -CCT TAG AGT AAA TCC AGT GG-30 50 -TCC CGA ATG TGC CCA ACT TC-30 50 -GAA AGA TCT GCC GAA AGT AAG-30 50 -CGT GGA CAT CTT CAG GGC C-30 50 -GTC GTG TTC AAT GCC ACC G-30 50 -AAG ACC AGG GCG AGT ACA AG-30 50 -AGC GAC ATG AAC CAA GGT TTC-30 50 -ATC GTT TAG CTT CGT GTT CGC-30 50 -CTT TCT TTT ACC ACA CGG TTG-30 50 -TCA GTC ATG AAG GCT TGC GTA-30 50 -TCG CAC ACA TTG GCA TCC ATT G-30
SeRelish SeApolipophorin III SeTransferrin-1 SeTransferrin-2 SeAttacin-A SeAttacin-B SeGloverin SeHemolin SeCecropin
2. Materials and methods
SeGallerimycin
2.1. Insect and fungus cultures The beet armyworm, S. exigua, used in this study was originated from a field population infesting welsh onion (Allium fistulosum) in Andong, Korea. The larvae were reared on an artificial diet (Goh et al., 1990) at 25 °C and the adults were fed 10% sucrose. An entomopathogenic fungus, Beauveria bassiana, was cultured on solid potato dextrose agar (PDA) medium (5% potato extract, 0.5% dextrose, 1.7% agar) at 25 °C for 7 days. The cultured fungal colonies were resuspended with phosphate buffered saline (PBS, 50 mM phosphate, 0.7% NaCl, pH 7.0). Mixture of conidia and mycelia was filtered using a filter paper (pore size = 20 lm) and the resulting flow-through suspension was used for further study. 2.2. Enzyme activity measurement PLA2 activity was measured by spectrofluorometry using a pyrene-labeled phospholipid [1-hexadecanoyl-2-(1-pyrenedecanoyl)sn-glycerol-3-phosphatidyl choline] as a substrate in the presence of bovine serum albumin (Radvanyi et al., 1989). The fluorometric phospholipid was dissolved in ethanol to prepare 10 mM stock solution and 10% of bovine serum albumin (BSA) was prepared in sterilized distilled water. The reaction mixture (2 ml) was prepared in a cuvette by sequentially adding 1946 ll of 50 mM Tris–HCl buffer (pH 7.0), 20 ll of 10% BSA, 12 ll of 1 M CaCl2, and 20 ll of enzyme extract. The reaction was initiated by addition 2 ll of 10 mM pyrene substrate and subsequently fluorescence intensity was monitored with an Aminco Bowmen Series 2 luminescence spectrometer (FA257, Spectronic Instruments, USA) using excitation and emission wavelengths of 345 and 398 nm, respectively. The specific enzyme activity was calculated in pmol/min according to method of Radvanyi et al. (1989). For pH preference, different pH solutions were prepared with stock solutions of sodium phosphate buffer using Henderson–Hasselbalch equation. To check inhibition of its activity, 2 ll of test chemical was used in above described reaction mixture. 2.3. RT-PCR of different antimicrobial peptides (AMPs) Total RNA was isolated from larvae of S. exigua with Trizol reagent (Invitrogen, Carlsbad, CA, USA). First strand cDNA was synthesized with 1 ll of the extracted total RNA by using RT-Premix oligo-dT (50 -CCA GTG AGC AGA GTG ACG AGG ACT CGA GCT CAA GC TTT TTT TTT TTT TTT T-30 , Intron, Seoul, Korea). AMP genes were
amplified with gene-specific primers (Table 1). PCR was performed with 35 cycles of denaturation (94 °C, 1 min), annealing (50 °C, 1 min), and extension (72 °C, 1 min). As a control, expression of b-actin was analyzed with gene-specific primers (50 -TGG CAC CAC ACC TTC TAC TAC-30 and 50 -CAT GAT CTG GGT CAT CTT CT-30 ) to confirm the integrity of cDNA. 2.4. RNA interference (RNAi) Double stranded RNAs (dsRNAs) of SeToll, SeRelish and a viral gene ORF302 as a control dsRNA (Park and Kim, 2010) were prepared according to the manufacturer’s instruction of Megascript RNAi kit (Ambion, Massachusetts, TX, USA). Briefly, each gene fragment (342 bp for SeToll, 446 bp for SeRelish and 419 bp for ORF302) produced by PCR using each pair of the gene-specific primers was cloned into pCR2.1 vector (Invitrogen, Carlsbad, CA, USA). To screen the insert direction, plasmid DNAs containing recombinant SeToll, SeRelish and ORF302 were analyzed by restriction enzyme digestion using Sal I, Bam HI (Bioneer, Daejon, Korea) and Dde I (BEAMS Bio, Seoul, Korea), respectively. Sense and antisense strands were synthesized using T7 RNA polymerase at 37 °C for 4 h. To be effective, these dsRNAs were injected twice to S. exigua larvae. The first was at the 3 days old 4th instar (last day of this instar) and the second shot was at one day old 5th instar. dsRNA was prepared with Metafectene PRO (Biontex, Plannegg, Germany) in 1:1 volume ratio and incubated at 25 ± 1 °C for 20 min. Two l of the dsRNA (50 ng/ll) solution was injected into larval hemocoel at every time. Knockdown of specific mRNA was evaluated by RT-PCR every 12 or 24 h after the second injection of the dsRNA. 2.5. Nodule formation assay Nodulation assay was performed by injecting fungal suspension in a volume of 5 ll (5 104 conidia/larva) through an abdominal proleg using a 10 ll Hamilton syringe (Hamilton, Reno, Nevada, USA). After 8 h incubation at 25 °C, the treated larvae were dissected on dorsal side and the melanized nodules on its gut and fat body were initially counted under a stereoscopic microscope (SZX9, Olympus, Tokyo, Japan) at 50x magnification. After the alimentary canal was removed, nodules in the previously unexposed areas and remaining internal tissues were then counted and added to the initial count. Each treatment consisted of 10 test larvae.
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2.6. Data analysis Treatment means and variances were analyzed in one-way ANOVA by PROC GLM of SAS program (SAS Institute, 1989). All means were compared by least squared difference (LSD) tests at Type I error = 0.05. 3. Results 3.1. PLA2 activities of hemocyte and fat body are enhanced by a fungal infection Hemocyte and fat body are immune-associated tissues and analyzed in their PLA2 activity in S. exigua (Fig. 1). With increase of substrate concentration, specific PLA2 activities of these two tissues and plasma increased and reached their respective maximal levels, in which maximal enzyme activity of hemocyte PLA2 was greater than those of plasma and fat body (Fig. 1B). Based on these substrate-enzyme kinetic curves, Michaelis–Menten parameters of different PLA2s were estimated. Hemocyte PLA2 was significantly
Table 2 Enzyme kinetic parameters of PLA2s extracted from different tissues of fifth instar Spodoptera exigua. PLA2 source
Enzyme kinetic parametersA KM (lM)
Hemocyte Plasma Fat body
Vm (pmol/min/lg) a
0.34 ± 0.11 0.25 ± 0.05b 0.26 ± 0.09b
a
3.11 ± 0.23 2.06 ± 0.07b 1.79 ± 0.12b
I50 (nM) against BZAB 16.52 ± 3.71a 1.26 ± 3.19b 1.62 ± 6.81b
A Different letters followed by standard deviations are significantly different in means in each parameter at Type I error = 0.05 (LSD test). B Benzylideneacetone.
different from those of fat body and plasma in substrate affinity (KM) and maximal enzyme catalytic capacity (Vm) (Table 2). To determine the difference in PLA2s of different tissues, their susceptibilities to a specific PLA2 inhibitor were analyzed. Benzylideneacetone (BZA) is a specific PLA2 inhibitor derived from an entomopathogenic bacterium, X. nematophila, culture broth (Ji et al. 2004). It significantly inhibited PLA2 activities of all three tissue sources (Fig. 1B). Fat body and plasma PLA2s were more susceptible than that of hemocyte (Table 2). PLA2 activity increased in the immune-associated tissues, fat body and hemocyte, and in plasma after injection of B. bassiana (Fig. 2). The induction of PLA2 activity occurred as early as 2–4 h after the fungal infection. 3.2. RNAi of Toll signal suppresses specific AMP gene expressions and PLA2 activation
Fig. 1. PLA2 activities of the immune-associated tissues of fat body (FB) and hemolymph separated into hemocyte (HC) and plasma (PL) of fifth instar larvae of Spodoptera exigua. (A) Change in specific enzyme activities with increase of substrate concentration in different tissue type PLA2s. (B) Inhibition of PLA2 activities of different tissue type PLA2 by a specific PLA2 inhibitor, benzylideneacetone (BZA). Each measurement consisted of three independent replications. Different letters above standard deviation bars indicate significant difference among means at Type I error = 0.05 (LSD test).
To link immune signal pathways to PLA2 activation, RNAi was conducted against Toll and Imd pathways, respectively (Fig. 3). Both SeToll and SeRelish (a component of Imd pathway) were markedly expressed in response to bacterial immune challenges (Hwang, 2011). However, challenge with B. bassiana strongly induced expression of SeToll gene, but weakly induced expression of SeRelish. The expression of SeToll was specifically inhibited by dsRNA specific to SeToll, but not by dsRNA specific to SeRelish (Fig. 3A). Under theses RNAi conditions, we analyzed expressions of nine AMP genes in fat body and hemocyte (Fig. 3B). In both tissues, both attacins and cecropin were highly inducible to fungal infection, whereas gallerimycin, hemolin, and transferrin I were slightly inducible. However, apolipophorin III, gloverin, and transferrin II were strongly expressed irrespective of the fungal infection. When dsRNA specific to SeRelish or SeToll were respectively treated, these AMPs showed different suppression patterns. Apolipophorin III and transferrin II were insensitive to either dsRNAs due to their strong constitutive expressions. The other seven AMPs were inhibited in their expression by dsRNA specific to SeToll in both tissues. By contrast, dsRNA specific to SeRelish inhibited expression of only cecropin in hemocytes. Such inhibitory patterns were more evident in hemocytes than fat body. These results confirmed independent immune signaling pathways of Toll and Imd in S. exigua. Under these specific RNAi conditions, PLA2 activation of different tissues in response to fungal infection was analyzed (Fig. 4). dsRNA specific to SeToll significantly inhibited the activation of PLA2s of all tested tissues in response to the fungal infection. However, dsRNA specific to SeRelish did not suppress PLA2 activation in tested tissues. 3.3. Hemocyte nodulation is inhibited by dsRNA treatment specific to Toll pathway, but not to Imd pathway Eicosanoids mediate hemocyte nodule formation in response to bacterial or fungal infection (see Introduction). Inhibition of PLA2
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4. Discussion
Fig. 2. Induction of PLA2 activity in different PLA2s extracted from hemocyte (HC), plasma (PL), and fat body (FB). An entomopathogenic fungus, Beauveria bassiana, was hemocoelically injected into fifth instar larvae of Spodoptera exigua in a dose of 1.1 105 conidia and incubated for different periods at 25 °C. Each measurement consisted of three independent replications. Different letters above standard deviation bars indicate significant difference among means at Type I error = 0.05 (LSD test).
activation prevents eicosanoid biosynthesis, which would be expected to result in suppression of nodule formation. In response to fungal infection, S. exigua larvae significantly induced nodule formation compared to solvent injection (Fig. 5A). The nodule formation was significantly suppressed in the larvae treated with dsRNA specific to SeToll. By contract, dsRNA specific to SeRelish did not suppress the nodule formation. Exogenous AA addition significantly rescued nodule formation inhibited by RNAi of SeToll gene expression (Fig. 5B).
Eicosanoids mediate both cellular and humoral immune responses against various microbes (Stanley, 2005). Biosynthesis of eicosanoids is inducible to pathogen infection presumably after nonself recognition (Tunaz et al., 2003; Park and Kim, 2011). Depending on surface molecular structures, various microbes are discriminated by specific pattern recognition receptors (Medzhitov and Janeway, 2000). After recognition, two main immune signaling pathways, Toll and Imd pathways, lead to expression of various AMP genes depending on microbes (Lemaitre and Hoffmann, 2007). Thus, eicosanoid biosynthesis and Toll/Imd pathways may be associated after nonself recognition. This functional link was demonstrated in T. castaneum in response to various bacterial challenge, in which both Toll and Imd pathways are associated with PLA2 activation (Shrestha and Kim, 2010). This current study proved that only Toll signal pathway activated eicosanoid biosynthesis in response to a fungal infection in S. exigua. First, two main immune effectors (hemocyte and fat body) that possessed signal components of Toll/Imd pathways showed their specific PLA2 activities that were significantly induced by fungal infection. The activated PLA2 would catalyze AA release from phospholipids for subsequent eicosanoid biosynthesis. Second, the fungal infection induced expression of several AMPs that were mostly modulated by expression of Toll. The AMPs induced by Toll expression were distinct from those induced by Imd component (SeRelish). Third, RNAi experiments showed that dsRNAToll significantly inhibited activation of PLA2 in response to the fungal infection, but dsRNARelish did not. Fourth, hemocyte nodule formation induced by the fungal infection was significantly inhibited by dsRNAToll, but not by dsRNARelish. Lastly, the suppressed nodule formation by dsRNAToll was rescued by addition of AA. PLA2s constitute a superfamily of enzymes that catalyze phospholipid hydrolysis at the sn-2 ester bond and have been classified into at least 15 groups comprising of four main types: secretory (sPLA2), cytosolic Ca2+-dependent (cPLA2), cytosolic Ca2+-independent (iPLA2), and PAF acetyl hydrolase/oxidized lipid (LpPLA2) PLA2 (Burke and Dennis, 2009). Immune-associated PLA2s were detected from hemocyte, plasma, and fat body in S. exigua. These PLA2 activities were significantly enhanced by infection of B. bassiana. However, these different tissue PLA2s showed different enzyme kinetics, in which hemocyte PLA2 showed lower substrate affinity, higher catalytic capacity, and higher sensitivity to BZA (a specific PLA2 inhibitor) than plasma and fat body PLA2s. BZA is a competitive inhibitor against catalytic activity of PLA2 associated with insect immune responses (Shrestha and Kim, 2007; Kwon and Kim, 2008). Plasma and fat body PLA2s were not different with each other in the enzyme properties, such as KM, Vm, and inhibitor susceptibility. Recently four immune-associated PLA2s are identified in T. castaneum (Shrestha et al., 2010). All these PLA2s are secretory type and susceptible to BZA (Shrestha and Kim, 2010). Antibody raised from a T. castaneum sPLA2 did not reacted with PLA2 extracts of S. exigua (data not shown). This suggests that immune-associated PLA2s of S. exigua may have unique molecular forms. Also, the difference between hemocyte and other tissue PLA2s suggests that more than one type of PLA2s may play crucial role in immune responses in S. exigua. RNAi experiments suggest that Toll and Imd signal pathways act in immune responses of S. exigua. Toll and Imd immune signal pathways are well known in Drosophila (Lemaitre et al., 1995; Michel et al., 2001; Tanji et al., 2007). Upon infection of Gram-positive bacteria (Lys-type peptidoglycan) or fungi, Toll pathway is activated by prior proteolytic activation of proSpätzle in the plasma and its subsequent binding to the Toll receptor leads to an intracellular signaling cascade, which results in degradation of Cactus (an
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Fig. 3. RNA interference of immune signal genes (SeRelish and SeToll) suppresses gene expressions of different antimicrobial peptides (AMPs) in the fifth instar Spodoptera exigua in response to infection of Beauveria bassiana (Bb). Nine AMPs of S. exigua in this study included Apolipophorin III (SeApoLp III), attacin A (SeAtt A), attacin B (SeAtt B), cecropin (SeCec), gloverin (SeGlv), gallerimycin (SeGal), hemolin (SeHem), transferrin I (SeTf I), and transferrin II (SeTf II). b-Actin was used in this RT-PCR to confirm the integrity of cDNA preparation. (A) Specific interference of SeRelish or SeToll expressions by their specific double stranded RNAs (dsRNAs). A viral gene (ORF302) was used for dsRNA control. RT-PCRs were performed at 24 h after the dsRNA injections (200 ng per larva). (B) Inhibition of AMP gene expression by treatment of dsRNA specific to SeRelish or SeToll. At 24 h after dsRNA injection, larvae were individually injected with Bb in 5 104 conidia and incubated for 8 h at 25 °C. The treated larvae were then analyzed for AMP expressions by RT-PCR. Control represents a sham injection using PBS.
inhibitor of kB) and allows NF-jB (Dif or Dorsal) to migrate into nucleus to induce AMP gene expressions (Hoffmann, 2003). By contrast, Imd pathway is activated by the challenge of Gram-negative bacteria (diaminopimelate-type peptidoglycan), in which cooperative activity of PGRP-LC and PGRP-LE recognition receptors
activates unidentified transmembrane receptor and subsequent an adaptor protein, Imd, associates with FADD with its death domain (Leclerc and Reichhart, 2004). Thereafter a sequential activation of TAK1, Dredd, and IKK complex cleaves inactive NF-jB (Relish), which migrates into the nucleus to induce specific AMPs (Tanji
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Fig. 5. Hemocyte nodule formation in response to Beauveria bassiana (Bb) infection is linked to Toll signal pathway in Spodoptera exigua. At 24 h after dsRNA injection, larvae were individually injected with Bb in 5 104 conidia and incubated for 8 h at 25 °C. Each nodulation assay consisted of three independent replications. Different letters above standard deviation bars indicate significant difference among means at Type I error = 0.05 (LSD test). (A) Specific inhibition by treatment of dsRNA specific to SeToll. (B) Rescue effect on the suppressed nodulation by the addition of arachidonic acid (100 ng per larva). Control represents Bb injection.
Fig. 4. Induction of PLA2 activities of hemocyte (HC), plasma (PL), and fat body (FB) via Toll signal pathway in response to Beauveria bassiana (Bb) infection in fifth instar Spodoptera exigua. At 24 h after dsRNA injection, larvae were individually injected with Bb in 5 104 conidia and incubated for 8 h at 25 °C. Each enzyme activity measurement consisted of three independent replications. Different letters above standard deviation bars indicate significant difference among means at Type I error = 0.05 (LSD test).
and Ip, 2005). In response to B. bassiana, S. exigua larvae expressed all nine tested AMPs, in which except apolipophorin III and transferrin II that were constitutively expressed from a semi-RT-PCR analysis, seven AMPs were controlled under expression of SeToll expression because expressions of these seven AMPs were inhibited by dsRNAToll. However, the contribution of Imd pathway in the fungal infection was not much significant because itsnockdown by dsRNARelish inhibited expression of cecropin, but the AMP expression was also controlled by Toll pathway. These results suggest that Toll and Imd signal pathways independently mediate immune signals in S. exigua, in which Toll signal pathway mediates immune signal in response to infection of B. bassiana. Though PLA2s of three tissues were activated by infection of B. bassiana, dsRNAToll significantly inhibited the enzyme induction. By contrast, dsRNARelish did not inhibit the induction of PLA2 activity.
In addition, dsRNAToll significantly inhibited hemocyte nodule formation against the fungal infection, which was rescued by addition of AA. This suggests that downstream signal of Toll pathway may activate the immune-associated PLA2, which in turn activate biosynthesis of eicosanoids that mediate the cellular immune. Though little is known in cross-talk between Toll signal and PLA2 activation in invertebrates, mammalian immune cells show that PLA2 is activated by its phosphorylation due to kinases activated by Myd88 that is an adaptor molecule in a downstream of Toll-like receptor in response to immune challenge (Qi and Shelhamer, 2005). In T. castaneum, PLA2 was activated by de novo synthesis of PLA2 or by translocation to cell membrane (Shrestha and Kim, 2010), in which Toll signal may modulate both activations. These facts in other systems suggest that Myd88 may be an adaptor between Toll signal and PLA2 activation in S. exigua and that knockdown of Myd88 expression may uncouple the PLA2 activation by Toll signal pathway. Furthermore, in mammalian immune cells, Myd88 activates MAPKs, which subsequently activates cPLA2 by phosphorylation (Clark et al., 1991; Kramer et al., 1996). This suggests that there may be kinase(s) that is activated by Toll pathway and finally activates PLA2 of S. exigua. Our data provide the first insight of Toll immune signaling in its role other than AMP production in insects in response to fungal infection. Also, the specific coupling of Toll signaling and PLA2
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