three metabolites from an entomopathogenic bacterium, xenorhabdus nematophila, inhibit larval...

Insect Science (2011) 18, 282–288, DOI 10.1111/j.1744-7917.2010.01363.x

ORIGINAL ARTICLE

Three metabolites from an entomopathogenic bacterium,Xenorhabdus nematophila, inhibit larval development ofSpodoptera exigua (Lepidoptera: Noctuidae) by inhibitinga digestive enzyme, phospholipase A2

Jaehyun Kim and Yonggyun KimDepartment of Bioresource Sciences, Andong National University, Andong, Korea

Abstract An entomopathogenic bacterium, Xenorhabdus nematophila, has been knownto induce significant immunosuppression of target insects by inhibiting immune-associatedphospholipase A2 (PLA2), which subsequently shuts down biosynthesis of eicosanoids thatare critical in immune mediation in insects. Some metabolites originated from the bacterialculture broth have been identified and include benzylideneacetone, proline-tyrosine andacetylated phenylalanine-glycine-valine, which are known to inhibit enzyme activity ofPLA2 extracted from hemocyte and fat body. This study tested their effects on digestivePLA2 of the beet armyworm, Spodoptera exigua. Young larvae fed different concentrationsof the three metabolites resulted in significant adverse effects on larval development evenat doses below 100 μg/mL. In particular, they induced significant reduction in digestiveefficiency of ingested food. All three metabolites significantly inhibited catalytic activity ofdigestive PLA2 extracted from midgut lumen of the fifth instar larvae at a low micromolarrange. These results suggest that the inhibitory activities of the three bacterial metaboliteson digestive PLA2 of S. exigua midgut may explain some of their oral toxic effects.

Key words digestion, midgut, phospholipase A2, Spodoptera exigua, Xenorhabdus ne-matophila

Introduction

The bacterium, Xenorhabdus nematophila, is mutualis-tically associated with the entomopathogenic nematode,Steinernema carpocapsae (Akhurst, 1982). The bacteriaare localized in a specific site of the nematode gut anddelivered to lepidopteran hosts when the nematode en-ters the insect hemocoel (Forst et al., 1997). Within thehemocoel, the bacteria are released out of the nematodeand suppress insect immune responses by inhibiting en-

Correspondence: Yonggyun Kim, Department of Biore-source Sciences, Andong National University, Andong 760-749,Korea. Tel: +82 54 8205638; fax: +82 54 8206320; email:[email protected]

zyme activity of phospholipase A2 (PLA2), which sub-sequently shuts down production of eicosanoids (Park &Kim, 2000). Eicosanoids are a group of C20 polyunsatu-rated fatty acids oxidized from arachidonic acid, which isderived from phospholipid by a catalytic activity of PLA2

(Dennis, 1994). X. nematophila also produces protease II,which is capable of digesting cecropin antimicrobial pep-tide in Galleria mellonella and Pseudaletia unipunctata(Caldas et al., 2002), and secretes cytotoxic lipopolysac-charides to impair hemocytes to foster the host immuno-suppressive state (Dunphy & Webster, 1988).

Culture broth of X. nematophila possesses PLA2 in-hibitory activity against different PLA2 originating fromvarious biological sources (Park et al., 2004). The cul-ture broth has been further analyzed to purify thePLA2 inhibitor(s), which were then identified into one

C© 2010 The AuthorsJournal compilation C© Institute of Zoology, Chinese Academy of Sciences

282

Inhibition of digestive PLA2 by three bacterial metabolites 283

monoterpenoid and two peptide metabolites: benzylide-neacetone (BZA), proline-tyrosine (PY) and acetylatedphenylalanine-glycine-valine (Ac-FGV) (Ji et al., 2005;Shrestha et al., 2010a). As a kind of molecular targetfor the metabolites, immune-associated PLA2 genes havebeen identified in Tribolium castaneum (Shrestha et al.,2010b) and their recombinant enzymes have been in-hibited by the bacterial metabolites (Shrestha & Kim,2009). In an effort to develop novel insecticides usingthese metabolites, these bacterial metabolites have beenapplied along with Bacillus thuringiensis and show sig-nificant synergistic effect with the bacterial pesticide dueto their immune-suppressive effects in two lepidopteranspecies, Plutella xylostella and Spodoptera exigua (Kwon& Kim, 2007, 2008). BZA alone possesses antibacterialactivity against plant pathogens and acaricidal effect (Jiet al., 2005; Park et al., 2009).

Despite significant potential of these bacterial metabo-lites to be developed as novel insecticides, there is nodirect evidence on their independent toxic effect on insectpests. This study reports oral toxic effects of the three bac-terial metabolites on larvae of S. exigua. It also explainstheir oral toxic effects by analyzing their inhibitory effectson digestive PLA2 activity.

Materials and methods

Insect rearing

Beet armyworm, S. exigua, used in this study originatedfrom a field population infesting Welsh onion in Andong,Korea. The larvae were reared for ≈15 years in the labo-ratory on artificial diet (Gho et al., 1990) under 25 ± 1◦Cand 16 : 8 h (L : D). Adults were fed 10% sucrose.

Bioassay of effects of BZA, PY or Ac-FGV on larvaldevelopment of S. exigua

BZA (trans-4-phenyl-3-buten-2-one) was purchasedfrom Sigma-Aldrich, Korea (Seoul, Korea). PY and Ac-FGV were synthesized by Peptron, Inc. (Seoul, Korea). Allinhibitors were resuspended and diluted with dimethylsul-foxide (DMSO) (Sigma-Aldrich, Korea). Oral treatmentsused artificial diets containing different concentrations(10, 100 and 1 000 μg/mL) of BZA, PY or Ac-FGV. Toavoid any heat inactivation, the inhibitors were added tothe diet preparation after cooling down at ≈60◦C. Newlymolted second instar S. exigua larvae were individuallytreated with the diets until pupation. For the control,DMSO was added to the diet at the same concentrationas the metabolite treatment. Each treatment was inde-

pendently replicated three times. Each experimental unitconsisted of 30 larvae.

Efficiency of food utilization

Late fourth instar larvae of S. exigua were fed for3 days with artificial diet mixed with BZA, PY or Ac-FGV at 100 μg/mL. The weights of the larvae and theartificial diets were measured before and after the feed-ing duration. Weights of frass were also measured afterfeeding duration. Approximate digestibility (AD), effi-ciency of conversion of ingested food (ECI), and effi-ciency of conversion of digested food (ECD) were com-puted: AD (%) = ([weight of food ingested − weight offrass]/weight of food ingested) × 100, ECI (%) = (larvalweight gain/weight of food ingested) × 100, and ECD(%) = (larval weight gain/ [weight of food ingested −weight of frass]) × 100. Each treatment was independentlyreplicated three times. Each experimental unit consistedof 10 larvae.

Digestive enzyme extraction

Fifth instar S. exigua larvae grown on control artificialdiet were dissected to collect the midgut. Gut lumen con-tents were obtained by squeezing the midgut and werecentrifuged at 14 000 g for 5 min at 4◦C to obtain thesupernatant, which was used for luminal enzyme extract.The remaining gut tissue were washed with 100 mmol/Lphosphate buffer saline (PBS, pH 7.2) containing 0.7%NaCl and homogenized with phosphate buffered saline(PBS). After centrifugation at 14 000 g for 5 min at 4◦C,the supernatant was used for cellular enzyme extract.

For a treatment to analyze PLA2 activity in the gut lu-men after 48 h feeding of inhibitor-containing artificialdiets, the diets containing 100 μg/mL each of BZA, PYand Ac-FGV were prepared and treated to newly moltedfifth instar larvae. The luminal enzyme extract was pre-pared as described above.

Assessing PLA2 enzyme activity

PLA2 activity was determined by spectrofluorometryusing a pyrene-labeled phospholipid, 1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycerol - 3 - phosphatidylcholine(Molecular Probes, Eugene, OR, USA) as the substratein the presence of bovine serum albumin (BSA) (Rad-vanyi et al., 1989). The pyrene-labeled substrate was pre-pared at 10 mmol/L with 100% ethanol. The reaction mix-ture (2 mL) was prepared in a quartz cuvette by adding

C© 2010 The AuthorsJournal compilation C© Institute of Zoology, Chinese Academy of Sciences, Insect Science, 18, 282–288

284 J. Kim & Y. Kim

sequentially 50 mmol/L Tris buffer (pH 7.0), 10% BSA,1 mol/L CaCl2 and an enzyme preparation. The reactionwas initiated by addition of 10 mmol/L pyrene-labeledsubstrate and subsequently monitored for fluorescenceintensity in an Aminco Bowmen Series 2 luminescencespectrometer (FA257, Spectronic Instruments, Madison,WI, USA) using excitation and emission wavelengths of345 and 398 nm, respectively.

Data analysis

All studies were performed in three independent repli-cates and plotted by mean ± standard deviation (SD)using Sigma plot (Systat Software, Inc., Point Rich-mond, CA, USA). The means were compared by a leastsquares difference (LSD) test of one-way analysis of vari-ance (ANOVA) using PROC GLM of the SAS program(SAS Institute Inc., 1989) and discriminated at Type Ierror = 0.05.

Results

Oral toxic effect of BZA, PY or Ac-FGV on larvaldevelopment of S. exigua

To obtain any oral toxicity of three bacterial metabo-lites, they were mixed with the artificial diet of S. exiguain different concentrations. The second instar larvae werethen treated to these diets and their survival monitoreduntil pupation (Fig. 1). All three bacterial metabolitesgave increasingly significant adverse effects on larvaldevelopment with increase of their doses (F = 17.78;df = 3, 24; P < 0.000 1). These toxic effects were de-tected even below a dose of 100 μg/mL in all threemetabolites. However, there was significant differencein the inhibitory effects among inhibitors (F = 4.08;df = 6,24; P = 0.005 8). Among three metabolites, BZAshowed significantly higher toxicities than the others at10 and 100 μg/mL (F = 7.47; df = 1,12; P = 0.018 2).There was no significant difference in the oral toxici-ties between PY and Ac-FGV (F = 0.40; df = 1,24;P = 0.531 0).

The effects of the bacterial metabolites on develop-mental rate from larva to pupa were analyzed (Fig. 2).Newly molted second instar larvae were fed an artificialdiet containing 100 μg/mL of each bacterial metabolite.When the larval period was estimated from survivors, allthree bacterial metabolites significantly induced devel-opmental retardation of S. exigua larvae (F = 147.67;df = 3,8; P < 0.000 1). Among three metabolites, BZAwas significantly more inhibitory to larval development

Dose (μg/mL) in diet

Larv

al m

orta

lity

(%)

0

20

40

60

80

100

BZA PYAc-FGV

Control 10 100 1000

Fig. 1 Oral toxicities of three bacterial metabolites againstSpodoptera exigua. Second instar larvae were treated with artifi-cial diets containing different concentrations of the compounds.An experimental unit consisted of 30 larvae and treatments werereplicated three times. Larval mortality corrected by Abbott(1925) formula in each experimental unit was estimated fromfailure rate (%) of larval to pupal development (1 − [the num-ber of successful pupation/30]) × 100. The error bars indicatestandard deviations.

than the others (F = 106.91; df = 1,8; P < 0.000 1).There was no significant difference in the developmentrate between PY and Ac-FGV (F = 0.73; df = 1,8;P = 0.418 6).

Control PY Ac-FGV BZA

Larv

al p

erio

d (d

ays)

0

2

4

6

8

10

12

14

16a

bb

c

Fig. 2 Effects of developmental retardation on survivors treatedwith three bacterial metabolites in Spodoptera exigua. Newlymolted second instar larvae were treated with artificial dietscontaining 100 μg/mL of the compounds. Larval period wasmeasured from the second instar to pupation. An experimentalunit consisted of 30 larvae (survived until pupation) and eachtreatment was replicated three times. Different letters above thestandard deviation bars indicate significant differences amongmeans at Type I error = 0.05 (least significant difference test).



AD ECD ECI

0

10

20

80

100ControlPY Ac-FGVBZA

Dig

estib

ility

(%

)

ab b

a

b

c c

b

a

bc c

Fig. 3 Effects of three bacterial metabolites on efficiency offood utilization of Spodoptera exigua. The fourth instar larvaewere treated for 72 h with artificial diets containing 100 μg/mLof the compounds. Weight of diet ingested, weight of frass andweight gained, were measured from individual larvae. Each treat-ment consisted of 30 larvae. ‘AD’, ‘ECD’ and ‘ECI’ representapproximate digestibility, efficiency of conversion of digesteddiet and efficiency of conversion of ingested diet, respectively.Different letters above standard deviation bars indicate sig-nificant differences among means in each category at Type Ierror = 0.05 (least significant difference test).

Adverse effects of BZA, PY or Ac-FGV on digestionefficiency of S. exigua

To explain the oral toxicities of the bacterial metabo-lites, their effects on digestion efficiency were analyzedusing three different parameters (Fig. 3). Fourth instar lar-vae were fed an artificial diet containing 100 μg/mL ofeach bacterial metabolite. There was no significant dif-ference between control and treatments in approximatedigestion (AD) efficiency except Ac-FGV treatment. Theincrease of AD in Ac-FGV treatment appeared to becaused by increase of ingested food amount. However,the conversion efficiencies of ingested (ECI) or digested(ECD) food were significantly low in all treated larvaecompared to that in control larvae (F = 6.96; df = 3,8;P = 0.012 8 for ECI and F = 6.67; df = 3,8; P = 0.014 4for ECD).

Inhibitory effects of BZA, PY or Ac-FGV on digestivePLA2 of S. exigua

Digestive enzymes were extracted from midgut lumenand its epithelial cells, respectively, to test whether bac-terial metabolites inhibited digestive PLA2, which ledto malfunctioning in digestion (Fig. 4A). All three bac-

(A)

(B)

0 10 100 1000

Rel

ativ

e P

LA2

activ

ity (

%)

30

40

50

60

70

80

90

100

110

30

40

50

60

70

80

90

100

BZAPYAc-FGV

Lumen

Cellular

BZAPYAc-FGV

PLA

2 ac

tivity

(pm

ol/ m

in/μ

L)

0

2

4

6

8

10

PY BZA Ac-FGVControl

a

cd

b

Fig. 4 Inhibitory effects of three bacterial metabolites on di-gestive phospholipase A2 (PLA2) of the fifth instar larvae ofSpodoptera exigua. (A) In vitro enzyme inhibition assay. PLA2

was extracted from the midgut lumen and epithelial cells. Theenzyme extracts (100 μg) were incubated with different concen-trations of the metabolites for 10 min at 25◦C and added withpyrene-labeled substrate to measure residual enzyme activities.(B) Residual PLA2 activities in midgut lumen after 48 h feed-ing of artificial diet containing 100 μg/mL of each bacterialmetabolite. Each treatment was independently replicated fivetimes. The error bars indicate standard deviations.

terial metabolites gave significant inhibitory effects onPLA2 activity in a dose-dependent manner (F = 4 431.87;df = 3,24; P < 0.000 1 for luminal extract andF = 2 295.39; df = 3,24; P < 0.000 1 for cellular extract).


286 J. Kim & Y. Kim

There was also significant variation in the PLA2 inhibitionamong three bacterial metabolites in luminal (F = 62.56;df = 2,24; P < 0.000 1) and cellular (F = 1 577.48;df = 2,24; P < 0.000 1) extracts. In particular, in cellularPLA2 extract, Ac-FGV was not as potent as the others(F = 3 108.28; df = 1,24; P < 0.000 1).

Based on the in vitro inhibitory activities of the bacterialmetabolites against digestive PLA2 extracts, we measuredmidgut PLA2 activities of the larvae feeding on the treatedartificial diets (Fig. 4B). Compared to control, the larvaefeeding on the treated diets had significantly lower en-zyme activities (F = 385.07; df = 1,8; P < 0.000 1).Among inhibitor treatments, BZA was more potent thanthe others in inhibitory effects (F = 64.53; df = 1,8;P < 0.000 1).

Discussion

In an effort to develop novel insecticides, three metabo-lites (BZA, PY and Ac-FGV) originated from X. ne-matophila were analyzed in this study to determine theiradverse effects on development of S. exigua. The bacterialmetabolites were identified to inhibit immune-associatedPLA2 of S. exigua through a series of chemical fraction-ations from the culture broth of X. nematophila (Ji et al.,2005; Shrestha et al., 2010a). Thus the bacterial metabo-lites significantly increased pathogenicity of Bacillus thu-rigiensis (Bt) against two lepidopteran insects, Plutellaxylostella and S. exigua (Kwon & Kim, 2007, 2008). Thisstudy tested any toxic effects of these metabolites againstlarval development without any help from Bt pesticide.All three bacterial metabolites gave significant oral toxic-ities in dose-dependent manners to immature developmentof S. exigua, although they were different in inhibitory ef-fects. BZA was more potent in inducing developmentalretardation and inhibiting pupal development than the oth-ers. A similar tendency was also observed in earlier studies(Park et al., 2009; Seo & Kim, 2009), in which BZA wasalso more effective to synergize Bt pathogenicity and toinhibit growth of plant bacterial pathogens. This toxicityvariation among these metabolites can be explained bytheir affinity differences in targeting PLA2 active sites(see below).

The oral toxicities of the metabolites were explainedby their interferance with the digestive efficiency of S.exigua by inhibiting catalytic activity of digestive PLA2.All three metabolites of X. nematophila adversely affectedthe digestive efficiency of S. exigua. Except for Ac-FGV,these bacterial metabolites were not different from thecontrol treatment in AD, suggesting that these metabolitesmight not significantly interfere with feeding activity of

S. exigua larvae. The increase of AD in Ac-FGV may beexplained by the increase in amount of feeding to compen-sate for poor digestibility. However, all these metabolitessignificantly reduced both ECI and ECD, suggesting in-efficiency in conversion of ingested food to weight gain,probably due to malfunction in digestion due to inhibitoryactions of the bacterial metabolites to digestive PLA2 ofS. exigua.

In general, various PLA2s are made from a singlegene family and are largely classified into secretoryPLA2 (sPLA2), cytosolic and calcium-independent PLA2

(iPLA2), and cytosolic and calcium-dependent PLA2

(cPLA2), which are characterized by molecular sequence,structure and localization (Schaloske & Dennis, 2006).In insects, only sPLA2 have been known from Apis mel-lifera, Drosophila melanogaster, and T. castaneum withtheir catalytic activities (Dudler et al., 1992; Ryu et al.,2003; Shrestha et al., 2010b). Compared to bee venomsPLA2, four TcsPLA2s are known to play a role in im-mune responses (Shrestha et al., 2010b) and are inhib-ited by BZA as well as bromophenacyl bromide (BPB),a specific sPLA2 inhibitor (Marchi-Salvador et al., 2009;Shrestha & Kim, 2009). Several digestive PLA2s havebeen biochemically analyzed in insects. A digestive PLA2

was characterized in Cicindella circumpicta and showedcalcium-dependency and its expression depended on feed-ing activity (Uscian et al., 1995). Another digestive PLA2

extracted from the midgut of Aedes aegypti also showedassociation with feeding activity (Nor Aliza & Stan-ley, 1998). A lepidopteran digestive PLA2 originatingfrom the midgut contents of Manduca sexta also showedfeeding-associated enzyme activity (Rana et al., 1998;Rana & Stanley, 1999). Although yet unidentified in theirmolecular structures, insect digestive PLA2s may be in-cluded in sPLA2 just like mammalian digestive PLA2sand have been postulated to play a critical role in diges-tion by providing lysophospholipids, as well as digestionof polyunsaturated fatty acids linked to the sn-2 position(Stanley, 2006a). In insects which do not have bile saltfor digestion, lysophospholipids liberated from phospho-lipids by catalytic activity of PLA2 may function as a bilesalt to facilitate lipid digestion (Stanley, 2006b). Threemetabolites of X. nematophila showed inhibitory activityagainst digestive PLA2 of S. exigua, which may criticallyimpair lipid digestion of ingested food and cause devel-opmental retardation and fatality. Interestingly, all threemetabolites share a common chemical structure of ben-zylpropane, any alteration of which causes significant lossof their inhibitory effects (Shrestha et al., 2010a). Con-sidering that the core structure is shared also with BPB,it may interact with active sites of PLA2. The differen-tial inhibitory activities to target PLA2 may be caused by



variation in molecular motifs around the core structure.Subsequently, the variation in PLA2 inhibition may resultin differential oral toxicity in these bacterial metabolites.

Acknowledgments

Most of this work was supported by AGEND 2009 re-search program of Rural Development Administration,Suweon, Korea. J. Kim was supported by the second stageBK21 program of the Ministry of Education, Science andTechnology, Seoul, Korea.

References

Abbott, W.S. (1925) A method of computing the effectivenessof an insecticide. Journal of Economic Entomology, 18, 265–267.

Akhurst, R.J. (1982) Antibiotic activity of Xenorhabdus spp.,bacteria symbiotically associated with insect pathogenic ne-matodes of the families Heterorhabditidae and Steinerne-matidae. Journal of General Microbiology, 128, 3061–3065.

Caldas, C., Chergui, A., Pereira, A. and Simoes, N. (2002)Purification and characterization of an exracellular proteasefrom Xenorhabdus nematophila involved in insect immuno-suppression. Applied and Environmental Microbiology, 68,1297–1304.

Dennis, E.A. (1994) Diversity of group types, regulation andfunction of phospholipase A2. Journal of Biochemistry, 269,13057–13060.

Dudler, T., Chen, W.Q., Wang, S., Schneider, T., Annand, R.R.,Dempcy, R.O., Crameri, R., Gmachl, M., Suter, M. and Gelb,M.H. (1992) High-level expression in Escherichia coli andrapid purification of enzymatically active honey bee venomphospholipase A2. Biochimica et Biophysica Acta, 1165, 201–210.

Dunphy, G.B. and Webster, J.M. (1988) Lipopolysaccharidesof Xenorhabdus nematophila (Enterobacteriaceae) and theirhaemocyte toxicity in non-immune Galleria mellonella (In-sect: Lepidoptera) larvae. Journal of General Microbiology,134, 1017–1028.

Forst, S., Dowds, B., Boemare, N. and Stackebrandt, E. (1997)Xenorhabdus and Photorhabdus spp.: Bugs that kill bugs.Annual Review of Microbiology, 5, 47–72.

Gho, H.G., Lee, S.G., Choi, K.M. and Kim, J.H. (1990) Simplemass-rearing of beet armyworm, Spodoptera exigua (Hubner)(Lepidoptera: Noctuidae), on an artificial diet. Korean Jour-nal of Applied Entomology, 29, 180–183.

Ji, D., Yi, Y., Kang, G.H., Choi, Y.H., Kim, P., Baek, N.L. andKim, Y. (2005) Identification of an antibacterial compound,benzylideneacetone, from Xenorhabdus nematophila against

major plant-pathogenic bacteria. FEMS Microbiology Letters,239, 241–248.

Kwon, S. and Kim, Y. (2007) Immunosuppressive action of pyro-proxyfen, a juvenile hormone analog, enhances pathogencityof Bacillus thuringiensis subsp. Kurstaki against diamond-back moth, Plutella xylostella (Lepidoptera: Yponomeuto-dae). Biological Control, 42, 72–76.

Kwon, B. and Kim, Y. (2008) Benzylideneacetone, an im-munosuppressant, enhances virulence of Bacillus thuringien-sis against beet armyworm (Lepidoptera: Noctuidae). Journalof Economic Entomology, 101, 36–41.

Marchi-Salvador, D.P., Fernandes, C.A.H., Silveira, L.B.,Soares, A.M. and Fontes, M.R.M. (2009) Crystal structure of aphospholipase A2 homolog complexed with p-bromophenacylbromide reveals important structural changes associated withthe inhibition of myotoxic activity. Biochimica et BiophysicaActa, 1794, 1583–1590.

Nor Aliza, A.R. and Stanley, D.W. (1998) A digestive phospholi-pase A2 in larval mosquitoes, Aedes aegypti. Insect Biochem-istry and Molecular Biology, 28, 561–569.

Park, Y. and Kim, Y. (2000) Eicosanoids rescue Spodopteraexigua infected with Xenorhabdus nematophilus, the sym-biotic bacteria to the entomopathogenic nematode Stein-ernema carpocapsae. Journal of Insect Physiology, 46, 1469–1476.

Park, Y., Kim, Y. and Stanley, D. (2004) The bac-terium Xenorhabdus nematophila inhibits phospholipase A2

from insect, prokaryote, and vertebrate sources. Naturwis-senschaften, 91, 371–373.

Park, Y., Kim, M., Lee, G., Chun, W., Yi, Y. and Kim, Y. (2009)Inhibitory effects of an eicosanoid biosynthesis inhibitor, ben-zylideneacetone, against two spotted spider mite, Tetrany-chus urticae, and a bacterial wilt-causing pathogen, Ralsto-nia solanacearum. Korean Journal of Pesticide Science, 13,185–189.

Radvanyi, F., Jordan, L., Russo-Marie, F. and Bon, C. (1989)A sensitive and continuous fluorometric assay for phospholi-pase A2 using pyrene-labeled phospholipids in the presenceof serum albumin. Analytical Biochemistry, 177, 103–109.

Rana, R.L. and Stanley, D.W. (1999) In vitro secretion of di-gestive phospholipase A2 by midgets isolated from tobaccohornworms, Manduca sexta. Archives of Insect Biochemistryand Physiology, 42, 179–187.

Rana, R.L., Sarath, G. and Stanley, D.W. (1998) A digestivephospholipase A2 in midguts of tobacco hornworms, Mand-uca sexta. Journal of Insect Physiology, 44, 297–303.

Ryu, Y., Oh, Y., Yoon, J., Cho, W. and Baek, K. (2003) Molec-ular characterization of a gene encoding the Drosophilamelanogaster phospholipase A2. Biochimica et BiophysicaActa, 1628, 206–210.

SAS Institute, Inc. (1989) SAS/STAT User’s Guide, release 6.03ed. SAS Institute, Cary, NC.


288 J. Kim & Y. Kim

Schaloske, R.H. and Dennis, E.A. (2006) The phospholipase A2

superfamily and its group numbering system. Biochimica etBiophysica Acta, 1761, 1246–1259.

Seo, S. and Kim, Y. (2009) Two entomopathogenic bacteria,Xenorhabdus nematophila K1 and Photorhabdus temper-ata subsp. temperata ANU101 secrete factors enhancing Btpathogenicity against the diamondback moth, Plutella xy-lostella. Korean Journal of Applied Entomology, 48, 385–392.

Shrestha, S., Hong, Y.P. and Kim, Y. (2010a) Two chem-ical derivatives of bacterial metabolites suppress cel-lular immune responses and enhance pathogenicity ofBacillus thuringiensis against the diamondback moth,Plutella xylostella. Journal of Asia Pacific Entomology, 13,55–60.

Shrestha, S. and Kim, Y. (2009) Biochemical characterizationof immune-associated phospholipase A2 and its inhibition by

an entomopathogenic bacterium, Xenorhabdus nematophila.Journal of Microbiology, 47, 774–782.

Shrestha, S., Park, Y., Stanley, D. and Kim, Y. (2010b) Genesencoding phospholipase A2 mediate insect nodulation reac-tions to bacterial challenge. Journal of Insect Physiology, 56,324–332.

Stanley, D.W. (2006a) Prostaglandins and other eicosanoids ininsects: biological significance. Annual Review of Entomol-ogy, 51, 25–44.

Stanley, D.W. (2006b) The non-venom insect phospholipase A2.Biochimica et Biophysica Acta, 1761, 1383–1390.

Uscian, J.M., Miller, J.S., Sarath, G. and Stanley-Samuelson,D.W. (1995) A digestive phospholipase A2 in the tiger bee-tle, Cicindella circumpicta. Journal of Insect Physiology, 41,135–141.

Accepted April 22, 2010


three metabolites from an entomopathogenic bacterium, xenorhabdus nematophila, inhibit larval...

Documents