Phenoloxidase Activity in Hemolymph of Naive and Beauveriabassiana-Infected Spodoptera exigua Larvae

S. Y. HUNG AND D. G. BOUCIAS

Entomology & Nematology, Building 970, Hull Road, Gainesville, Florida 32611

Received April 24, 1995; accepted August 15, 1995

The majority of Spodoptera exigua hemolymph phe-noloxidase (PO) activity is localized in the hemocytesof naive larvae. Mid sixth instar larvae possessed 0.2 ±0.5 and 3.4 ± 2.6 units (1 unit = D0.001 Å/min at 490 nm)of PO activity/ml of hemolymph equivalent in theplasma and hemocyte lysate (HL) fractions, respec-tively. The S. exigua HL PO had a pH optimum of 7.0when assayed against DL-Dopa. This enzyme is presentin hemocytes as a proenzyme: activation of PO couldbe inhibited with either 10 mM diisopropyl fluorophos-phate or soybean trypsin inhibitor (1 mg/ml). The rela-tive activity of HL PO was not inhibited by the addi-tion of EGTA or EDTA. The PO titers in HL and plasmafractions sampled from sixth instar larvae during thefeeding period (0–60 hr) of the sixth larval instar werenot significantly different. However, a 10 × increase inPO activity was observed in plasma sampled from thewandering and prepupal stages. A series of assays wasconducted to monitor the PO activity in Beauveriabassiana-infected S. exigua larvae. At 24 hr postchal-lenge the PO titers in plasma and HL fractions frominfected larvae were comparable to PO titers in frac-tions from saline controls. Analysis of PO activities re-vealed that by 48–60 hr postchallenge the PO titers inthe HL sampled from infected larvae decreased seven-fold, whereas plasma PO titers increased. The in-creased titer of PO in the plasma did not inhibit fungaldevelopment. © 1996 Academic Press, Inc.

KEY WORDS: Beauveria bassiana; Spodoptera exigua;phenoloxidase activity; insect immunity; hemocyte bi-ology.

INTRODUCTION

Phenoloxidase (EC 1.14.18.1) (PO), also referred toas tyrosinase, is commonly found in nature and cata-lyzes the hydroxylation of monophenols, then oxidizesthe resulting o-diphenols to quinone intermediates(Sugumaran, 1990). Quinones are subsequently con-verted to melanin or react with proteins forming pro-tein–catechol complexes. In arthropods the products ofthe phenoloxidase cascade are believed to be involved

in wound healing, in sclerotization of cuticle, and in therecognition and melanization of foreign particles (forreview see Söderhäll, 1992). The PO detected in bothcuticle and hemolymph is derived from a proenzyme,prophenoloxidase (proPO). Activation of proPO in cer-tain hosts is mediated by a Ca2+-dependent serine pro-tease cascade which in turn may be activated by vari-ous microbial cell wall components (lipopolysaccharide,laminarin, zymosan, etc.; Söderhäll and Smith, 1986).Endogenous activators (such as serine proteases) de-tected in the insect cuticle (Saul and Sugumaran, 1987)are believed to activate PO production during thewound repair reaction.The concept that PO is an important component of

the internal defense is based on numerous observa-tions of non-self being coated with a melanized layer(Götz and Vey, 1986). Certain classes of insect hemo-cytes recognize and bind to foreign particles and re-cruit additional hemocytes which encapsulate and pro-duce a melanized coat. Activation of PO results in theproduction of ‘sticky’ proteins which have been impli-cated in the recognition of non-self (Söderhäll et al.,1979). For example, Leonard et al. (1985b) was able toshow that activation of the PO cascade affiliated withmonolayers of Galleria mellonella hemocytes enhancedthe phagocytic activity of the cells. Whether the PO isserving as the opsonin under in vivo conditions is un-clear. For example, certain insects such as the Dro-sophila melanogaster mutant strains, deficient in PO,are still able to successfully recognize and encapsulatenon-self (Rizki and Rizki, 1990). In addition to poten-tially serving in nonspecific recognition processes, me-tabolites associated with the PO cascade (quinones,melanin) are toxic. Söderhall and Ajaxon (1982) and St.Leger et al. (1988), using in vitro assay methods, dem-onstrated that PO-derived quinones or melanin werefungistatic/fungicidal. Other investigators have sug-gested that the PO products produced during the invivo encapsulation reaction may suppress microbialgrowth but are not always lethal (Götz and Vey, 1986).In spite of the high titer of proPO associated with in-sect hemolymph, many parasites and pathogens suc-

JOURNAL OF INVERTEBRATE PATHOLOGY 67, 35–40 (1996)ARTICLE NO. 0006

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0022-2011/96 $18.00Copyright © 1996 by Academic Press, Inc.

All rights of reproduction in any form reserved.

cessfully develop in the hemocoel and kill host insects.Microbes, such as the bacteria affiliated with entomog-enous nematodes, are able to survive exposure to thepro PO → PO cascade, and their presence in the hemo-lymph eventually suppresses PO activity (Dunphy andWebster, 1984). Similarly, several endoparasitoids, in-cluding Cotesia glomerata and Hyposoter exigua, havebeen reported to inhibit both the encapsulation re-sponse and PO activity of host insects (Kitano et al.,1990; Stoltz and Cook, 1986).In the following study we have examined the PO ac-

tivity in larval hemolymph of the beet armywormSpodoptera exigua larvae. In our laboratory, S. exiguahas served as a model host to study the in vivo devel-opment of the mycopathogen Beauveria bassiana(Hung and Boucias, 1992; Hung et al., 1993; Mazet etal., 1994). In this report, the characteristics of PO inthe hemolymph of naive S. exigua larvae were exam-ined during its last larval instar. Second, we examinedthe effects of the vegetative development of B. bassianaon the PO associated with S. exigua hemolymph.

MATERIALS AND METHODS

Preparation of Hemolymph Samples forPhenoloxidase Assay

Beet armyworm, Spodoptera exigua, eggs were ob-tained from cultures maintained at the USDA/ARSlaboratory (Gainesville, FL). Larvae were reared on asemisynthetic insect diet (Greene et al., 1976) at 26°Cunder a 16 hr light: 8 hr dark photoperiod. Initial stud-ies involved assaying the PO activity in the hemo-lymph of mid sixth instar larvae. Individual larvaewere initially surface-sterilized with 95% ethanol andrinsed 2× in sterile distilled H2O. Hemolymph sampleswere collected from puncture wounds on the secondproleg and 40 ml was mixed with an equivalent volumeof Hepes buffered saline (HBS) at 4°C. One half of thissample was used to measure PO activity in whole he-molymph. The second aliquot (40 ml) was centrifuged(12,000g) at 4°C for 5 min to pellet the hemocytes.Plasma was transferred immediately to new tubes andhemocyte pellets resuspended in 40 ml of HBS. Hemo-lymph, plasma, and hemocyte fractions were homog-enized on ice for 1 min with a microprobe sonicating tipfitted to a Sonic Dismembrator Model 300 (Fisher Sci-entific). Resulting cell debris was removed by centrifu-gation (12,000g) at 4°C for 5 min. Additional hemo-lymph samples were collected from three groups ofsixth instar larvae. Pooled hemolymph samples werediluted in HBS and the samples of hemolymph,plasma, and hemocyte lysate (HL) supernates wereprepared according to the protocols described for indi-vidual hemolymph samples. In all cases the sampleswere assayed immediately for PO activity.

Phenoloxidase Assay

The phenoloxidase assay was performed in a 96-wellmicrotiter plate. To each well, 200 ml of substrate, 40mM 2-methyl-3(3,4-dihydroxyphenyl)-DL-alanine (DL-DOPA), dissolved in HBS, pH 7.0, was added to 20 ml ofenzyme preparation. The absorbance at 490 nm wasmeasured using a MR600 microplate reader (DynatechLaboratories, Inc.). PO activity was expressed as units/ml hemolymph, and specific activity of PO was definedas units/mg protein. One unit represents D0.001Å at490 nm/min. The BCA protein assay (Pierce ChemicalCo.) was used to determine protein concentration usingbovine serum albumin as the protein standard.

pH Optima

Hepes buffers with pH ranging from 4 to 9 were usedto determine the optimal pH for S. exigua hemolymphPO. Hemolymph from sixth instar larvae was pooledand mixed gently with an equivalent volume of HBS at4°C. Aliquots (100 ml) of each hemolymph pool weretransferred into individual microcentrifuge tubes. Af-ter centrifugation at 12,000g at 4°C for 5 min, plasmawas removed, and hemocyte pellets were resuspendedin 100 ml of HBS with designated pH. PO samples wereprepared and enzymatic activities were assayed usingthe methods described above. Three replicate hemo-lymph pools were used as a source for the HL samplesused in these assays.

Effect of EDTA, EGTA, STI, and DFP on thePO Activity

The effect of ethylenediaminetetraacetic acid(EDTA), ethylene glycol-bis (b-aminoethyl ether)N, N,N8, N9-tetraacetic acid (EGTA), diisopropyl fluorophos-phate (DFP), and soybean trypsin inhibitor (STI) onPO activity was tested by adding chemicals to bothsample and substrate buffers. The pH of the amendedbuffer solutions were adjusted to pH 7.0. Hemolymphfrom five sixth instar larvae was pooled on a paraffinmembrane, and 40-ml aliquots were mixed with either40 ml 2× EDTA (20 mM), EGTA (20 mM), DFP (2 and 20mM), or STI (0.2 and 2.0 mg/ml HBS) in microcentri-fuge tubes. HL and plasma samples were prepared us-ing the protocols described above, and resulting super-nates were used for PO assay with substrate-amendedbuffers.

Effect of B. bassiana Infection on the PO Activity

The effect of B. bassiana infection on the PO activitywas determined by measuring the PO activity inplasma and HL samples from sixth instar larvae in-fected with B. bassiana. To produce a synchronous in-fection, late fifth instar S. exigua larvae were injectedwith either 5 ml of 0.85% saline with or without 500 B.bassiana blastospores, according to the methods re-

HUNG AND BOUCIAS36

ported previously (Hung and Boucias, 1992). At 24, 48,and 60 hr after injection, 20 ml hemolymph from indi-vidual larvae was sampled and mixed with 20ml Hepessaline buffer. HL and plasma samples were preparedusing the methods described above, and supernateswere used for PO assay.

RESULTS

The majority of PO activity detected in hemolymphof sixth instar larvae was associated with the HL (Fig.1). The low levels of PO detected in the plasma of naivelarvae are believed to be artifacts of collection and han-dling of hemolymph samples. The optimal pH of POactivity in S. exigua HL samples was pH 7.0 using 40mM DL-DOPA as the substrate (Fig. 2). Below pH 6.0and above pH 8.0 the relative PO activity detected inHL preparations was suppressed. At pH 4.0 no PO ac-tivity was detected in the HL preparations.The PO associated with S. exigua larval hemocytes is

present in the cells as proPO. The baseline-specific ac-tivity of PO of HL samples from sixth instar larvaeprepared in HBS buffer averaged 1271 units/mg pro-tein (Table 1). The addition of either soybean trypsininhibitor (1 mg/ml) or DFP (1, 10 mM), both potent

serine protease inhibitors, significantly suppressed theproPO → PO activation in the HL preparations. Theaddition of the chelating agents, EDTA or EGTA, at 10mM, did not suppress the PO activity, suggesting thatthe ProPO activation process was not dependent on theavailability of divalent cations. Interestingly, bothEGTA and EDTA amendments caused slight increased

FIG. 1. The kinetics of phenoloxidase activity present in wholehemolymph, plasma, and hemocyte lysates prepared from individualSpodoptera exigua sixth instar larval hemolymph. One unit of phe-noloxidase represents D.001Å at 490 nm/min.

FIG. 2. pH optimum for the phenoloxidase activity associatedwith the Spodoptera exigua sixth instar hemocyte lysates.

TABLE 1The Effects of Various Inhibitors on the proPO→ PO Acti-

vation in Spodoptera exigua Hemocyte Lysate Preparationsa

Resulting PO activity (units/mg protein)b

Treatment (Mean ± SD)

Control 1271.6 ± 268.5bcEDTA (10 mM) 1738.5 ± 460.9aEGTA (10 mM) 1616.5 ± 524.3abSTI (0.1 mg/ml) 1041.6 ± 169.6cSTI (1 mg/ml) 523.1 ± 74.3dDFP (1 mM) 432.1 ± 125.1dDFP (10 mM) 287.6 ± 74.8d

a A total of eight different pools of S. exigua hemolymph werecollected and tested against the seven treatments (50 ml/hemocytelysate). Hemocyte lysates were prepared and assayed against DL-Dopa.

b Unit of phenoloxidase defined as D.001 Å at 490 nm/min. Thedata were analyzed using the Means–Tukey statement of the generallinear program (SAS Institute, 1985). Mean values ± SD for treat-ments having the same letter are not significantly different (a 40.05).

EFFECT OF Beauveria ON PHENOLOXIDASE ACTIVITY 37

levels of PO, suggesting these compounds may haveassisted in the activation of PO during sample prepa-ration.During the sixth larval instar, the relative levels of

PO activity in both plasma and HL fractions remainedconstant during the larval feeding period (0–60 hr).Hemocyte lysates contained approximately 4.0 unitsversus plasma preparations which contained 0.3 unitsper microliter hemolymph (Table 2). Due to high levelsof protein in the plasma fractions (z30 mg/ml), thespecific PO activity (units/mg protein) in the HLsamples were more than 100-fold greater than thosecalculated for plasma fractions. As the sixth instar lar-vae entered the ‘wandering phase’ a significant in-crease in PO activity was observed in the plasma frac-tions. However, the PO levels in HL preparationssampled during the wandering pupal stage were notdifferent from the high titers observed during the feed-ing phase (0–60 hr).The wound response of S. exigua larvae to injection

of saline did not cause significant alteration in the lev-els or distribution of hemolymph PO (Table 3). The POactivities in both plasma and HL samples variedamong the sixth instar larvae. However, in all larvaethe vast majority of the detectable PO resided in thehemocytes. Challenging S. exigua larvae with 500 blas-tospores of B. bassiana caused significant changes inthe hemolymph PO activity. The 24 hr postchallengeplasma samples from infected larvae contained four- tofivefold more PO than samples from saline-challengedlarvae. By 60 hr postchallenge, the PO titer in theplasma fraction of B. bassiana hemolymph, althoughless than detected in 24-hr samples, were significantlygreater than the PO titers detected in saline-injectedlarvae. Both the 48- and 60-hr plasma samples from B.bassiana-infected larvae contained active PO and pro-duced melanin when stored for a short duration on iceor at room temperature. Alternatively, the plasma ex-

tracted from saline-challenged larvae could be storedfor several hours prior to eliciting any detectable mela-nization reaction. The vegetative development of B.bassiana in the hemocoel of S. exigua caused dramaticalterations in PO levels associated with the HLsamples. By 48 hr postchallenge, the specific PO activ-ity associated with the infected HL samples was z20%of that detected in either that 24 hr B. bassiana or 48hr saline-injected HL samples. The combined specificPO activity of the 48 hr-infected plasma and HLsamples was 284 units versus 1159 units detected incomparable samples from saline-injected larvae. Thesedata suggest that the PO released from the hemocytesof infected larvae is either short-lived or is rapidly com-plexed to either host tissues or to the vegetative hyphalbodies. At the late stage of hyphal body development(60 hr) the PO levels in HL were dramatically sup-pressed, whereas the PO activity in the plasma fractionwas higher than the naive plasma preparations. Thedevelopment of B. bassiana in the hemocoel did notcause significant change in either the protein concen-trations of the plasma or HL samples.

DISCUSSION

Previous studies have demonstrated that the loca-tion of PO is dependent on the insect species. In Man-duca sexta and Sarcophaga bullata it has been re-ported that the proPO is in the plasma fraction (Sauland Sugumaran, 1987, 1988). Alternatively, inBlaberus craniifer and G. mellonella the proPO is lo-calized in the hemocytes (Schmitt et al., 1977; Leonardet al., 1985a,b). In S. exigua larvae the vast majority ofdetectable PO was in the HL samples. Potentially, theplasma fraction of S. exigua contained endogenous in-hibitors (protease inhibitors) which preventedproPO → PO activation (Saul and Sugumaran, 1988).As the S. exigua sixth instar larvae entered the non-

TABLE 2Relative Protein Levels and Phenoloxidase Activities in Hemolymph Samples Prepared from Naive S. exigua during Sixth

Instar to the Prepupal Stage

Plasma Hemocyte lysate

PO activity (units/ PO activity (units/ PO activity (units/ PO activity (units/Samplea N Proteinb mg protein)c ml hemolymph)c Protein mg protein) ml hemolymph)

24 hr 14 28.6 ± 6.0c 9.8 ± 22.4b 0.4 ± 0.6c 3.6 ± 1.0b 1091.9 ± 527.4a 4.2 ± 2.3ab48 hr 21 32.6 ± 8.6bc 5.3 ± 15.7b 0.2 ± 0.5c 3.8 ± 0.9b 1027.6 ± 804.8a 3.4 ± 2.6b60 hr 21 37.3 ± 7.2b 5.0 ± 10.1b 0.2 ± 0.5c 4.1 ± 1.0b 1096.1 ± 550.0a 4.2 ± 1.8abWandering 21 36.6 ± 10.0a 69.2 ± 42.2a 2.3 ± 1.2b 3.0 ± 0.8c 1059.7 ± 584.7a 4.3 ± 1.6abPrepupal 21 54.9 ± 10.0a 79.2 ± 24.5a 4.2 ± 1.0a 4.8 ± 1.2a 1079.4 ± 303.6a 5.5 ± 1.6a

a The 24, 48, and 60 hr represent the time post sixth instar larval molt. Wandering stage designates fully mature sixth instar whichstopped feeding and have begun to search for sites for the pupal molt. Prepupa are those wandering larvae which have both voided gutcontents and have burrowed into forming pupal site.

b Protein concentration (mg/ml) estimated using BCA assay (Pierce Chemical Co.) calibrated with BSA standards.c Phenoloxidase activity is presented as units (D.001 Å at 490 nm/min) per microliter of hemolymph equivalent. Data from plasma and

hemocyte lysates were analyzed separately using Means–Tukeys statement or the general linear program (SAS Institute, Inc. 1985). Meanvalues having the same letter are not significantly different at a 4 0.05.

HUNG AND BOUCIAS38

feeding wandering stage and constructed a pupal cell,the PO levels in the plasma fractions increased withoutnoticeable decreases in the PO titer in HL samples.Potentially, this increase could be due to either a re-duction in endogenous inhibitor activity, the synthesisof higher titers of PO, and/or an alteration in the he-mocyte sensitivity resulting in the release and activa-tion of the proPO during bleeding.Experiments demonstrated that the PO activity as-

sociated with S. exigua could be inhibited by the addi-tion of serine protease inhibitors which are believed toblock the activation of the proPO to PO. In severallepidoptera species, including G. mellonella, M. sexta,and Heliothis zea, amendment of hemolymph prepara-tions with such inhibitors has been demonstrated toinhibit PO activity (Leonard et al., 1985b; Aso et al.,1985; Lockey and Ourth, 1992). In certain systems theaddition of proteases such as trypsin, chymotrypsin,thermolysin, and subtilisin have been reported to acti-vate the proPO present in plasma samples (Suguma-ran, 1990). However, amendment of S. exigua plasmafractions with either trypsin, chymotrypsin, elastase,or subtilisin did not result in increased plasma PO ti-ters (data not shown), suggesting that S. exiguaplasma lacks endogenous proPO. Alternatively, the S.exigua plasma may contain high levels of endogenousprotease inhibitors or the proPO present in S. exiguaplasma requires a combination of activators as re-ported for the proPO of Hylaphora cercropia, whichrequires a C1 factor and a serine protease (Anderson etal., 1989). The PO activity present in S. exigua HLsamples did not require Ca2+ ions for activation andwas not inhibited by either EGTA or EDTA. In otherinsect systems, both EDTA and EGTA have been dem-onstrated to be potent inhibitors (Ashida and Söderh-all, 1984; Leonard et al., 1985a,b; Dunphy, 1991) and

are believed to prevent Ca2+-mediated activation of theserine proteases responsible for proPO→PO cascade.The PO in S. exigua HL had a pH optima (7.0) similarto that calculated for H. cercropia and G. mellonella(Anderson et al., 1989; Dunphy, 1991). Interestingly,buffers having a pH < 6.0 caused a dramatic reductionin activity, with no PO activity being detected at pH4.0. The sensitivity of the S. exigua PO to low pH mayexplain the inactivation of PO in hemolymph collectedin the acidic anticoagulant buffers used by Mead et al.(1986).Infection of S. exigua with B. bassiana caused

marked alterations in the levels and distribution of POactivity. Developmental studies of this mycopathogenin S. exigua have demonstrated that within 24 hr post-challenge the fungus undergoes limited growth and ispresent in low numbers (Hung and Boucias, 1992).However, at 24 hr, B. bassiana causes a significantsuppression in the ability of hemocytes to spread overa substrate (Hung et al., 1993). This alteration is be-lieved to be caused by metabolites which destabilizethe cytoskeleton of the insect hemocytes (Mazet et al.,1994). These ‘altered’ hemocytes at the 24 hr samplinginterval contain PO levels comparable to the naive he-mocytes. These results suggest that the hemocyte dys-function induced by B. bassiana is not simply an in-duced degranulation event as reported for certain ar-thropods challenged with various microbial cell wallpreparations (Söderhäll, 1992). The injected fungusfrom 24–60 hr postinjection undergoes extensive veg-etative replication as hyphal bodies in the host hemo-coel (Hung and Boucias, 1992). During this exponentialgrowth phase the fungus causes a dramatic reductionin the numbers and types of circulating hemocytes. Thedecrease in the PO titers of HL samples and the con-comitant increase in PO levels in plasma fractions dur-

TABLE 3Relative Levels of Detectable Protein and Phenoloxidase Activity in S. exigua Hemolymph Sampled during the in Vivo

Development of B. bassiana

Hemocyte lysate

PO activity (units/c PO activity (units/ PO activity (units/ PO activity (units/Samplea Proteinb mg protein) ml hemolymph) Protein mg protein) ml hemolymph)

24 hr saline 28.4 ± 6.9b 12.0 ± 23.7b 0.5 ± 0.9bc 3.3 ± 0.7ab 1576.9 ± 814.7ab 4.9 ± 2.1a48 hr saline 34.0 ± 6.9ab 12.0 ± 25.8b 0.4 ± 0.8bc 3.4 ± 1.4a 1147.0 ± 821.8a 4.3 ± 2.9a60 hr saline 34.5 ± 10.2ab 0.8 ± 1.6c 0.03 ± 0.04c 3.7 ± 1.2a 933.5 ± 665.4a 3.3 ± 2.1a

24 hr B. bassiana 28.0 ± 6.9a 70.7 ± 75.8a 2.0 ± 2.2a 2.6 ± 0.8bc 1148.3 ± 1154.1a 3.6 ± 2.8a48 hr B. bassiana 38.9 ± 8.6a 32.9 ± 31.4b 1.3 ± 1.3ab 2.5 ± 0.7c 251.4 ± 127.1b 0.7 ± 1.3b60 hr B. bassiana 35.8 ± 7.7a 33.4 ± 35.0b 1.2 ± 1.1ab 3.0 ± 1.1abc 132.7 ± 127.1b 0.4 ± 0.4b

a Insect larvae were injected as late fifth instar with 500 fungal cells or with the sterile saline, and after 24, 48, and 60 hr at 26°C individualhemolymph samples were taken and processed for phenoloxidase determination. A total of 21 individual larvae were sampled for eachtreatment.

b Protein concentration (mg/ml) estimated using BCA assay (Pierce Chemical Co.), using bovine serum albumin as standard protein.c Unit of phenoloxidase (PO) equals D0.001 Å at 490 nm/min. Data from plasma and hemocyte lysates were analyzed separately and the

average values ± SD having the same letter were not significantly different at a 4 0.05 (Tukeys HSD).

EFFECT OF Beauveria ON PHENOLOXIDASE ACTIVITY 39

ing this interval are believed to be due to the cytolyticeffects of B. bassiana on host hemocytes. The fate of the‘released’ proPO during this stage is unknown. The ob-served increase in the plasma PO activity did not in-terfere with the growth and development of B. bassi-ana hyphal bodies. At the interval when the circulatinghyphal bodies undergo a transition to the mycelialphase (50–72 hr postchallenge) the total phenoloxidaseactivity detected in the hemolymph is 50% less thanthat observed in naive larvae.

ACKNOWLEDGMENTS

The ARS/USDA laboratory (Gainesville, FL) is acknowledged forkindly providing the S. exigua eggs used in these experiments. Sup-port for this research was in part provided by a USDA competitivegrant (93-01815). Florida Experiment Station Journal No. R04507.

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