Journal of

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Journal of Invertebrate Pathology 95 (2007) 119–124

INVERTEBRATE

PATHOLOGY

Eicosanoid biosynthesis inhibitors increase the susceptibility ofLymantria dispar to nucleopolyhedrovirus LdMNPV

David Stanley a,*, Martin Shapiro b

a USDA/Agricultural Research Service, Biological Control of Insects Research Laboratory, 1503 Providence Rd., Columbia, MO 65203, USAb USDA/Agricultural Research Service, Beltsville, MD 20705, USA

Received 26 September 2006; accepted 6 February 2007Available online 22 February 2007

Abstract

Eighteen pharmaceutical inhibitors of eicosanoid biosynthesis were tested for their effects on gypsy moth, Lymantria dispar and itssusceptibility to the nucleopoly-hedrovirus LdMNPV. None of the inhibitors tested had any detrimental effects upon larval growthand development. Treatment with nine inhibitor/NPV combinations (e.g., bromophenacylbromide, clotrimazole, dexamethasone, escu-letin, flufenamic acid, indomethacin, nimesulide, sulindac, tolfenamic acid) resulted in 3.5- to 6.6-fold reductions in LC50s. Larvae treatedwith several other COX inhibitors did not yield significant LC50 reductions. We infer that eicosanoids act in insect defense responses toviral infection. Eicosanoids may act at three levels of insect immune reactions to viral infection, organismal (febrile response), cellular(hemocytic microaggregation, nodulation and plasmatocytes spreading reactions) and intracellular level (mechanisms responsible forinsect permissiveness to viral replication).Published by Elsevier Inc.

Keywords: Baculoviruses; Gypsy moth; Insect susceptibility; Eicosanoids; Non-steroidal anti-inflammatory drugs

1. Introduction

Insect susceptibility or nonsusceptibility to different par-asites and pathogens is dependent on several factors. Onthe basis of work with the tobacco hornworm Manduca

sexta, Stanley-Samuelson et al. (1991) suggested that cellu-lar actions accountable for clearing bacteria from hemo-lymph circulation were mediated by eicosanoids.Eicosanoids are oxygenated metabolites of certain C20polyunsaturated fatty acids, including arachidonic acid(Stanley, 2000, 2006; Stanley and Miller, 2006). Stanleyand colleagues identified two specific cellular actions, mic-roaggregation and nodule formation, which are mediatedby eicosanoids in tobacco hornworms (Miller et al., 1994).

Eicosanoids also mediate other aspects of cellularimmune reactions to bacterial infection, including cell

0022-2011/$ - see front matter Published by Elsevier Inc.

doi:10.1016/j.jip.2007.02.002

* Corresponding author. Fax: +1 573 875 5364.E-mail address: stanleyd@missouri.edu (D. Stanley).

spreading and in some species activation of prophenoloxi-dase (Mandato et al., 1997; Miller, 2005). The results ofexperiments with several other insect species indicated thateicosanoids mediate cellular immune reactions to bacterialinfections in over 20 insect species representing six orders(Stanley, 2006; Stanley and Miller, 2006).

Eicosanoids also mediate cellular immune reactions toparasitoid eggs (Carton et al., 2002), to infection by theinsect pathogenic fungi Metarhizium anisopliae (Deanet al., 2002) and Beauveria bassiana (Lord et al., 2002), aswell as protozoan infection by Trypanosoma rangeli (Gar-cia et al., 2004). Taken along with the work on cellularreactions to bacterial challenge, these findings support theview that eicosanoids mediate cellular reactions to bacte-rial, fungal, protozoan and parasitoid attack.

Most recently, in their work with larvae of the waxmoth, Galleria mellonella, Buyukguzel et al. (2007)reported that eicosanoids mediate nodule formation reac-tions to infection with the vertebrate virus, Bovine herpes

simplex virus-1. This finding with a vertebrate virus led us

120 D. Stanley, M. Shapiro / Journal of Invertebrate Pathology 95 (2007) 119–124

to pose the hypothesis that prostaglandins (PGs) or othereicosanoids are responsible for mediating insect defensereactions to baculoviruses (BVs). BVs are infectious toinsects and some other arthropods and some BV-insectrelationships are very specialized. To test our hypothesis,we recorded the influence of pharmaceutical inhibitors ofeicosanoid biosynthesis on larvae of the gypsy mothLymantria dispar and their susceptibility to a nucleopolyhe-drovirus LmMNPV.

2. Materials and methods

2.1. Insect colony

The New Jersey strain of Gypsy moth, L. dispar

(USDA-APHIS, Otis ANGB, MA) was used for all exper-iments. Larvae were reared on a wheat germ-based dietunder the conditions described by Bell et al. (1981).

2.2. Chemicals

The following chemicals (pharmaceutical inhibitors ofeicosanoid biosynthesis in mammals) were obtained fromSigma–Aldrich, St. Louis, MO: acetaminophen (0.066 M;CAS 103-90-2), acetyl salicylic acid (0.055 M; CAS 50-782), bromophernacyl bromide (0.036 M; CAS 93-73-0),clotrimazole (0.033 M; CAS 23593-75-1), dexamethasone(0.025 M; CAS 50-02-2), diclofenac (0.031 M; CAS15307-79-6), esculetin (0.056 M; CAS 05-01-1), fenoprofen(0.019 M; CAS 53746-45-5), flufenamic acid (0.036 M;CAS 530-78-9), ibuprofen (0.048 M; CAS 15687-27-1),indomethacin (0.028 M; CAS 53-86-1), ketorolac(0.027 M; CAS 74103-07-1), meclofenamic acid (0.031 M;CAS 6385-02-0), naproxen (0.040 M; CAS 26159-34-2),nimesulfide (0.032 M; CAS 51803-78-2), sulindac(0.028 M; CAS 38194-50-2), tolfenamic acid (0.038 M;CAS 13710-19-5), tolmetin (0.032 M; CAS 64490-92-2).All chemicals were diluted in distilled water. Not all ofthese chemicals are completely soluble in water, in whichcase a suspension of the chemicals was applied to the dietsurface.

2.3. Virus and test protocol

The gypsy moth nucleopolyhedrovirus virus (isolateLDP-226; US Forest Service, Hamden, CT) was used inall experiments. Occlusion bodies (OB) were extracted fromvirus-killed larvae using standardized methods (Shapiroet al., 1981). The insects were homogenized (e.g., each gramof larval tissue was blended in 9 g distilled water) and fil-tered through coarse cheesecloth. The filtrate was collected(=stock virus suspension) and 1 ml of the stock suspensionwas diluted in 9 ml distilled water. A sample was removedby Pasteur pipette and the concentration of this suspension(1:10) was determined using a double-line hemacytometerwith improved Neubauer ruling and phase contrast optics(430· magnification).

Dilutions were made from the stock suspension eitherin distilled water (standard treatment) or in a chemical(test treatment) to produce concentrations ranging from10 to 1 million OBs/ml and 1 ml of a virus suspensionwas placed onto the diet surface (180-ml container;4770 mm2 = surface area). Appropriate controls (e.g., dis-tilled water, test chemicals) were used. Ten second instars(7 d old; average weight, 30 mg per larva) were placed ineach 180-ml container and maintained for 21 d at 29 �C,50% RH and a photoperiod of 12:12 (L/D). Tests wererepeated 10 times with 30 larvae per virus dilution treat-ment and 30 control larvae (distilled water, test chemi-cals) per replicate. Mortality was recorded at day 7and every other day until day 21, when tests wereterminated.

2.4. Statistical methods

Concentration-mortality regressions were calculated todetermine the effects of selected pharmaceutical treatmentson LdMNPV activity. Slopes and LC50s were obtainedwith the probit option of POLO (LeOra Software, 1987).Means were separated for significance according to Fish-er’s protected least significant difference (LSD) test atP 6 .05 (Steel and Torrie, 1960).

3. Results

3.1. Effects of eicosanoid biosynthesis inhibitors on activity

of the gypsy moth NPV

Non-steroidal anti-inflammatory drugs (NSAIDs) sharethe common pharmaceutical action of cyclooxygenase(COX) inhibition. In our experiments, the virulence ofLdMNPV challenge increased significantly in insects trea-ted with some, but not all, COX inhibitors (Table 1). Con-trol experiments established the LC50 of LdMNPVchallenge (=virus only) at about 17.4 OB/mm2. LC50

values for insects treated with virus/flufenamic acid, virus/indomethacin, virus/tolfenamic acid, virus clotrimazole,and virus/diclofenac combinations were reduced 3- to 6-fold compared to virus only. We recorded relatively smallchanges in LC50 values with insects treated with othervirus/NSAID combinations, including virus/ibuprofen.Two other COX inhibitors, nimesulfide and suldinac, arespecific to COX-2 enzymes. Treatment with these chemi-cal/virus combinations yielded 4- to 5-fold reductions ofLdMNPV LC50. Treatment with the specific lipoxygenase(LOX) inhibitor, esculetin, in a virus/esculetin combina-tion, resulted in a 6-fold reduction in LC50. Finally, treat-ment with two phospholipase A2 (PLA2) inhibitors,bromophenacyl bromide and dexamethasone, resultedin � 3- to 5-fold reductions in LdMNPV LC50 values(Table 1).

Background control experiments demonstrated thatnone of the chemicals adversely affect larval growth anddevelopment.

Table 1Median lethal concentration (LC50) of LdNPVa with and without theaddition of eicosanoid inhibitors infecting larvae of Lymantria dispar

Treatment

Concentration (M) LC50

(95% CL)bActivityratioc

LdNPV/H2O 17.4 (9.6–30.1) 1.0

LdNPV plus

NSAID COX inhibitors

Flufenamic acid 0.036 2.6 (1.9–3.7)* 6.6Clotrimazole 0.033 2.9 (2.0–4.5)* 6.1Indomethacin 0.028 3.8 (2.4–6.2)* 4.6Tolfenamic acid 0.038 3.9 (2.3–6.4)* 4.5Diclofenac 0.031 5.4 (2.9–9.8) 3.2Tolmetin 0.032 7.8 (4.6–12.8) 2.2Ketorolac 0.027 9.7 (3.8–22.3) 1.8Meclofenamic acid 0.031 10.4 (7.3–14.7) 1.7Naproxen 0.040 10.4 (7.0–15.4) 1.7Acetaminophen 0.066 11.4 (10.0–13.0) 1.5Fenoprofen 0.019 12.1 (8.7–17.1) 1.4Ibuprofen 0.048 14.2 (9.9–21.0) 1.2Acetyl salicylic acid 0.055 16.5 (9.9–29.0) 1.1

NSAID COX-2

inhibitors

Nimesulide 0.032 4.1 (2.1–7.6)* 4.2Sulindac 0.028 5.0 (3.3–7.7)* 3.5

LOX inhibitor

Esculetin 0.056 6.2 (4.2–9.2)* 3.8

PLA2 inhibitors

Bromophenacylbromide

0.036 3.4 (3.0–3.9)* 5.1

Dexamethasone 0.025 3.7 (2.2–8.9)* 4.7

a LdMNPV was diluted in distilled water or in a NSAID. No larvalmortality was observed among larvae exposed to any of the chemicals.

b LC50s are expressed as OBs per mm2; 10 replicates; five virusconcentrations per treatment per replicate; total = 1500 larvae pertreatment.

c Activity ratios are calculated by dividing the LC50 for LdMNPV aloneby LC50s for each LdMNPV/inhibitor combination.

D. Stanley, M. Shapiro / Journal of Invertebrate Pathology 95 (2007) 119–124 121

4. Discussion

This research is a logical extension from prior findingsthat (1) eicosanoids occur in insects (Stanley-Samuelson,1987, 1991; Pedibhotla et al., 1997), (2) eicosanoids areinvolved in many insect physiological functions (Loheret al., 1981; Toolson et al., 1994; Stanley, 2006), and (3)eicosanoids have been implicated in insect immuneresponse to bacteria (Miller et al., 1994; Jurenka et al.,1997; Park and Kim, 2000; Park et al., 2003), fungi (Lordet al., 2002; Dean et al., 2002), parasitoids (Carton et al.,2002) and protozoa (Garcia et al., 2004). In order to deter-mine whether eicosanoids influence virus infection andlethality in gypsy moth larvae, we tested well-knownCOX-1 and COX-2 inhibitors, a LOX inhibitor and inhib-itors of PLA2. The outcomes of our experiments indicatethat treatments with many, but not all, pharmaceuticalinhibitors of eicosanoid biosynthesis led to sharply reducedLC50 values. Our data indicate that pharmaceutical inhibi-

tion of eicosanoid biosynthesis in gypsy moth larvae ren-ders them less able to protect themselves from viralchallenge. We infer that eicosanoids influence insect protec-tive reactions to viral challenge.

Our experimental design is grounded in the understand-ing that pharmaceutical eicosanoid biosynthesis inhibitorsfrom the biomedical arena similarly inhibit eicosanoid bio-synthesis in insects. This is certainly true for houseflies(Wakayama et al., 1986), house crickets (Murtaugh andDenlinger, 1982), Australian field crickets (Tobe andLoher, 1983) and tobacco hornworms (Stanley-Samuelsonand Ogg, 1994). Murtaugh and Denlinger (1982) reportedthat dietary COX inhibitors reduced PG levels in crickettissues. The studies with field crickets and tobacco horn-worms were conducted with in vitro enzyme preparations.Wakayama et al. (1986) found that in vitro housefly prepa-rations were sensitive indomethacin, naproxen, acetamino-phen and aspirin these compounds did not influence PGbiosynthesis after rearing the fly larvae on diets supple-mented with the same compounds. We recently found thatindomethacin inhibited cellular immune reactions toBHSV-1 when administered by injection and by feeding.The single study of the pharmacology of a COX inhibitorin insects, using tobacco hornworms, indicates that indo-methacin is taken up into all hornworm tissues, and thenexcreted in its unaltered form via Malpighian tubules afterabout 12 h. Once present in the frass, indomethacin wasmetabolized into more polar products, presumably by gutmicroflora (Miller and Stanley-Samauelson, 1996). Weinfer that eicosanoid biosynthesis inhibitors can be deliv-ered to insects by injection or per os in biologically mean-ingful ways.

A related issue is whether microbial infection stimulateseicosanoid biosynthesis. Tunaz et al. (2003) reported thatbacterial infection stimulates PLA2 activity in tobaccohornworm hemocytes. PLA2 is the first step in eicosanoidbiosynthesis. Also, Jurenka et al. (1999) showed that bacte-rial infection stimulated large increases in true armywormhemolymph titres of PGF2a Although such quantitativestudies have not been carried out with many insect species,the available data support the idea that infection stimulateseicosanoid biosynthesis, which can be inhibited followingpharmaceutical inhibitors.

The literature on the effects of PGs on mammalianviruses spans more than 30 years and indicates that PGsnot only act in mechanisms that inhibit viruses (Santoro,1997; Santoro et al. 1980, 1983; Pica et al. 2000) but canbe required for the reactivation (Gebhardt et al., 2005),replication (Ray et al., 2004; Liu et al., 2005; Waris andSiddiqui, 2005) or spread of viruses (Harbour et al.,1978). Eicosanoids inhibit mammalian virus replication inseveral DNA and RNA viruses in both in vivo andin vitro systems. For example, PGA and PGJ inhibit polio-virus replication in human HeLa cells (Conti et al., 1996).Similarly, PGA1 inhibits influenza virus replication inLLC-MK2 cells (Conti et al., 2001) and Mayaro virusreplication in Vero cells (Ishimaru et al., 1998; Burlandy

122 D. Stanley, M. Shapiro / Journal of Invertebrate Pathology 95 (2007) 119–124

and Rebello, 2001). PGA also inhibits Sendai virus replica-tion in African green monkey cells (Santoro et al., 1989),HIV-1 replication in human CEM-SS cells (Rozera et al.,1996), herpes simplex type 1 in human cornea stromal cells(O’Brien et al., 1996) and classical swine fever virus in PK15

cells (Freitas et al., 2003). With respect to insect cells, PGAalso inhibits vesicular stomatitis virus replication in Aedes

albopictus (Burlandy et al., 2004).On the other hand, Inglot and Woyton (1971) demon-

strated that NSAIDs, specifically efanamic acid and indo-methacin, were effective in the treatment of cutaneousherpetic infections. Subsequently, Wachsman et al. (1990)reported that such NSAIDS as ibuprofen and indometha-cin reduced the incidence of cutaneous, genital and oral-facial herpes virus. These early studies inferred that COXenzymes played an essential role for the herpes virus. In cellcultures, Harbour et al. (1978), Newton (1979), and Bakeret al. (1982) indicated that viral DNA synthesis, replica-tion, and spread increased in the presence of PGs. In thecase of herpes simplex virus type 1 and pseudorabies virusand hepatitis C virus, infection stimulated the induction ofCOX-2 but not Cox-1. When both COX enzymes wereinhibited by indomethacin (a non-specific COX-1 andCOX-2 inhibitor), NS398 (a specific COX-2 inhibitor) orFR122047 (a specific COX-1 inhibitor), virus yield wasgreatly reduced (Ray et al., 2004).

In the present study, a high proportion (=50%) of theinhibitors tested reduced the LC50 values of LdMNPV.These chemicals included inhibitors of COX-1 and COX-2enzymes (=flufenamic acid, indomethacin, tolfenamic acid),inhibitors of COX-2 (=nimesulide, sulindac), inhibitors ofLOX (=esculetin) as well as inhibitors of PLA2 (=bromoph-enacyl bromide) and provided inferential evidence of the roleof eicosanoids in viral pathogenesis. It might be thought thatthe results with inactive NSAIDs, those tested compoundsthat did not influence viral LC50s, argue against our hypoth-esis. We note an important point. In our experiments thepharmaceutical treatments were provided per os, rather thanby injection. In research into insects, most experiments usingNSAIDs as probes for eicosanoid biosynthesis have beendone using injection protocols. Such injections ensure thetreatment compounds are delivered to the hemocoel. Theper os treatments introduce a new layer of events that occurin the insect alimentary canal, such as high pH and differingmembrane transport properties, that can impact the effi-ciency of moving pharmaceuticals from the alimentary canalinto the hemocoels of experimental insects. In our view, theper os treatments provide new information that treatmentsat this level can effectively probe eicosanoid biosyntheticpathways in insects, although not all compounds are equallyactive. This comment highlights the point that for theseexperiments we used a single dosage for all compounds.Clearly, dose–response experiments will help determine dos-ages required for activity with the apparently inactive testpharmaceuticals.

A comprehensive model of eicosanoids actions in insecthost defense against microbial infection indicates these

actions take place at three levels of biological organization.At the organismal level, eicosanoids act in regulation ofbehavioral fever responses to microbial infection in locusts,Schistocerca gregaria (Bundey et al., 2003) and probablymost species. The fever response delayed the progress ofmycosis in treated locusts and reduced mortality due tothe pathogenic bacterium Serratia marcescens. At the cellu-lar level, eicosanoids mediate an unknown number ofhemocyte actions leading to observable hemocyte microag-gregation, nodulation and plasmatocyte elongation (Milleret al., 1994; Miller, 2005). Eicosanoids also act in variousintracellular events that regulate insect cell permissivenessto viral replication. We surfaced this point for mammaliancells just above. Goodman et al. (2006) reported thatin vitro virus production increased in a tobacco budworm,Heliothis virescens, cell line (HvAM1) after pretreatmentwith eicosanoid biosynthesis inhibitors and inferred thateicosanoids can influence host susceptibility at the cellularlevel. These data indicate that eicosanoids act in one ormore cell mechanisms involved in changing a cell line fromnon-permissive to semi-permissive to viral replication.

Insects express several defense reactions to viral chal-lenge, although these reactions probably do not representall insect anti-viral mechanisms. Apoptosis and midgut cellsloughing is one of the most extensively studied anti-viralreactions (Clem, 2005). Popham et al. (2004) suggested thatplasma phenoloxidase of larval lepidopterans (and perhapsall insects) act as an antiviral defense. Dietary phytochemi-cals mediate resistance to BV infection in some insect spe-cies. Hoover et al. (2000) reported that larvae of Heliothisvirescens reared on cotton were more resistant to baculovi-rus infection than larvae reared on lettuce. The authorsinferred that sloughing of infected midgut cells (a majormechanism of increasing resistance to baculoviral attack)took place at higher rates in larvae reared on cotton.

Two new anti-viral mechanisms underscore the futurepotentials of research in insect/virus interactions. Ponnuvelet al. (2003) isolated a lipase from the digestive fluids of thesilkworm Bombyx mori. The gene for this lipase, Bmlipase-

1, is expressed solely in midgut. The authors concluded thata digestive lipase may also act as a physiological deterrentto viral infection. More attention has been devoted to studyof anti-viral RNA silencing, which may occur in all eukary-otic cells (Saumet and Lecellier, 2006), although it may notbe so for mammalian cells (Cullen, 2006). The authors notethat specific silencing of infecting viral RNAs is inducednaturally in insect cells. The silencing pathways seem tobe very important because virally-encoded suppressors ofRNA silencing have evolved and are necessary for viralinfection (Li et al., 2002). We infer that eicosanoids act insome or all of these antiviral mechanisms.

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