Proc. Natl. Acad. Sci. USAVol. 92, pp. 10698-10702, November 1995Immunology

Role of the integument in insect defense: Pro-phenol oxidasecascade in the cuticular matrixMASAAKI ASHIDA* AND PAUL T. BREYtThe Institute of Low Temperature Science, Hokkaido University, Sapporo, Japan

Communicated by John H. Law, The University ofArizona, Tucson, AZ, July 31, 1995 (received for review June 9, 1995)

ABSTRACT The cuticle of the silkworm Bombyx mori wasdemonstrated to contain pro-phenol oxidase [zymogen ofphenol oxidase (monophenol, L-dopa:oxygen oxidoreductase,EC 1.14.18.1)] and its activating cascade. The activating cascadecontained at least one serine proteinase zymogen (latent form ofpro-phenol oxidase activating enzyme). When the extractedcascade components were incubated with Ca2+, the latent formof pro-phenol oxidase activating enzyme was itself activated and,in turn, converted through a limited proteolysis of pro-phenoloxidase to phenol oxidase. Immuno-gold localization of pro-phenol oxidase in the cuticle using a cross-reactive hemolymphanti-pro-phenol oxidase antibody revealed a random distribu-tion ofthis enzyme in the nonlamellate endocuticle and a specificorderly arrayed pattern along the basal border of the laminae inthe lamellate endocuticle of the body wall. Furthermore, pro-phenol oxidase was randomly distributed in the taenidial cush-ion of the tracheal cuticle. At the time of pro-phenol oxidaseaccumulation in the body wall cuticle, no pro-phenol oxidasemRNA could be detected in the epidermal tissue, whereasfree-circulating hemocytes contained numerous transcripts ofpro-phenol oxidase. Our results suggest that the pro-phenoloxidase is synthesized in the hemocytes and actively transportedinto the cuticle via the epidermis.

The chitinous exoskeleton of the insect is a nonliving matrix ofcarbohydrate and protein secreted from a monolayer of epi-dermal cells, which cover the entire surface of the insect,including respiratory tracheae, the anterior and posteriorportion of the digestive tract, and reproductive ducts (1). Thecuticle serves as a protective barrier between the internaltissues and the external environment. Injury causes the cuticleto darken or melanize around the damaged zone. Pasteur (2),while studying silkworm diseases, noted that the cuticles ofhealthy silkworms were often topically scratched, and as aresult the cuticle darkened around the injured area. Further-more, he observed that silkworms infected with the micro-sporidian Nosema bombysis often manifested a cuticular mel-anization reaction. Cuticular melanization is thought to be animportant defense reaction of insects because it is involved inwound healing and sequestration of pathogens (3). Morerecently it has been speculated that intermediate compounds(quinones) in the melanin synthesis from mono- or diphenolsare highly toxic to living cells, including infectious microor-ganisms (4).

Lai-Fook (5) studied the mechanism of the cuticular mel-anization reaction with regard to injury. She speculated thatphenol oxidase (monophenol, L-dopa:oxygen oxidoreductase,EC 1.14.18.1) was responsible for melanization and waspresent in a latent form, which in turn was activated by aproteinaceous activator upon injury. The regulation mecha-nism of the activation of pro-phenol oxidase (a zymogen ofphenol oxidase) and the site of its synthesis have remainedimportant unresolved questions in cuticular biochemistry for

nearly three decades (6). In this study we demonstrate thepresence of the pro-phenol oxidase cascade in the cuticle of thesilkworm.

MATERIALS AND METHODSOrganisms. Silkworms, Bombyx mori, were reared on the

artificial diet Silkmate 2M (Kyodo Shiryo, Tokyo), at 24°Cunder a 12-hr photoperiod. Experiments used fifth-instarlarvae reared to day 5 for pro-phenol oxidase cascade andlocalization studies. Fifth-instar larvae reared to day 2 wereused for Northern blot analysis.

Extraction of the Pro-phenol Oxidase Cascade from theCuticle. Twenty silkworm larvae were dissected in ice-coldacetate buffer 1 (50 mM acetate buffer at pH 5.2 containing 5mM EDTA). The integuments were then meticulously sepa-rated with a spatula to remove all tissues adhering to thecuticle. The tissue-free cuticles were subsequently rinsed twicein fresh acetate buffer I and then blotted with absorbent paperto remove excess liquid. Each cuticle was individually extractedwith 1 ml of acetate buffer II [same as acetate buffer I exceptcontaining 0.33 mM amidinophenylmethanesulfonyl fluoride(APMSF)] for 1-1.5 hr on ice. The extract was centrifuged at7000 x g for 10 min at 4°C. Combined supernatants (totalvolume of 18.5 ml) were then subjected to ion-exchangechromatography.Column Chromatography of Cuticular Extract on CM-

Toyopearl. To separate pro-phenol oxidase from its activatingcascade, cuticular extract was applied to a CM-Toyopearl(Tosoh, Tokyo) column (1.5 x 7 cm) that had been equili-brated with acetate buffer I, and the column was elutedsequentially with 60 ml of acetate buffer I and 50 ml of Trisbuffer I (10 mM Tris HCl containing 0.5 M KCI and 5 mMEDTA at pH 7.5). During the chromatography, the flow ratewas maintained at 2 ml/min, and effluent was monitored at280 nm using a UV-1 monitor (Pharmacia LKB). The flow-through fraction and the eluant corresponding to the first peakafter application of the Tris buffer I were saved. After their pHwas adjusted to 7.5 with 1 M Tris solution, both fractions wereindividually dialyzed against 1 liter of Tris HCl buffer II (10mM Tris HCI containing 0.1 mM EDTA at pH 7.5 at 23°C) for24 hr with one change of the buffer at 4°C. The dialyzedflow-through fraction (40 ml) and the first peak fraction (9.4ml) were referred to as the pro-phenol oxidase fraction andactivating cascade (AC) fraction, respectively. Protein concen-trations of the pro-phenol oxidase and AC fractions wereabout 370 ,ug/ml and 220 ,ug/ml, respectively.

Preparation of Pro-phenol Oxidase Activating Enzyme(PPAE) by Ca2+ Treatment of the AC Fraction and Its Inacti-vation by Treatment with Diisopropylfluorophosphate (DFP).Eighty millimolar CaC12 was added to the AC fraction to make

Abbreviations: APMSF, amindinophenylmethanesulfonyl fluoride;DFP, diisopropylfluorophosphate; AC, activating cascade; PPAE, pro-phenol oxidase activating enzyme.*To whom reprint requests should be addressed.tPresent address: Unite d'Ecologie des Systemes Vectoriels, InstitutPasteur, 25 rue du Dr. Roux 75724 Paris, Cedex 15, France.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

Proc. Natl. Acad. Sci. USA 92 (1995) 10699

its final concentration 4 mM and subsequently incubated on iceovernight. The incubated mixture was used as PPAE. Twomilliliters of PPAE was added to 100 ,ul of 1 M Tris HCl bufferat pH 8.6 to circumvent the drop in pH upon addition of DFP(final concentration of 20 mM). The mixture was incubated for 10hr and subsequently dialyzed against 1 liter of Tris buffer IIovernight at 4°C. In addition, the AC fraction was also treatedwith DFP prior to Ca2+ treatment.

Extraction of Cuticle with SDS and Urea. Cuticles wereobtained as described above and immediately frozen in liquidnitrogen. The tissue-free cuticles were ground to a fine powderin liquid nitrogen and immediately lyophilized without thaw-ing. The powder was homogenized in a glass homogenizer witha glass pestle (20 mg of powder per ml of 20 mM Tris HClcontaining 5 M urea, 5% SDS, and 3% 2-mercaptoethanol)followed by heating in boiling water for 3 min and then heldat 20°C for 12 hr. The homogenate was centrifuged at 15,000x g for 10 min. The supernatant was dialyzed against 20 mMTris HCl buffer (pH 7.2) containing 1% SDS and 1% 2-mer-captoethanol overnight. The dialyzed supernatant was used asan extract containing total extractable cuticular proteins.

Pro-phenol Oxidase and Anti-Pro-phenol Oxidase IgG.Pro-phenol oxidase was purified from larval hemolymph of B.mori as reported (7). The pro-phenol oxidase was dissolved inTris buffer II at a protein concentration of 100 ,ug/ml. Rabbitpolyclonal antibody was raised against homogeneous pro-phenol oxidase obtained from hemolymph of larval silkwormhemolymph, and anti-pro-phenol oxidase IgG was purifiedaccording to the method described before (8).

Colloidal Gold Immunocytochemistry for Localization ofPro-phenol Oxidase. All cuticle samples were excised in ice-cold10mM Tris-HCI buffer (pH 7.2) containing 664 ,uM APMSF andimmediately fixed in 3% (vol/vol) glutaraldehyde and embeddedin Lowicryl resin (8). Ultrathin sections were made using adiamond knife on several different body wall and tracheal integ-umental samples. Ultrathin sections were immunolabeled forpro-phenol oxidase using a polyclonal rabbit antisilkworm pro-phenol oxidase IgG (affinity purified). A goat anti-rabbit IgGconjugated to 10-nm colloidal gold was used to reveal the firstantibody (8). It was (H. I. Yamazaki and M.A., unpublishedobservation) shown that this antibody did not cross-react withcuticular laccase (p-diphenol: 02 oxidoreductase, E.C. 1.10.3.2,),which is involved in the sclerotization process (9). Control ex-periments were conducted in the same fashion except thatanti-pro-phenol oxidase IgG was substituted by the nonimmunerabbit IgG at the same concentration. Sections were viewed andphotographed at 80 kV on a JEOL model 100C electron micro-scope.SDS/PAGE and Immunoblotting of Pro-phenol Oxidase.

SDS/PAGE (10) was carried out in a 14% acrylamide gelexcept that N,N'-methylenebisacrylamide was replaced byAcrylAide crosslinker according to the manufacturer's (FMC)instructions. Immunoblotting of pro-phenol oxidase onto anImmobilon poly(vinylidene difluoride) membrane (Millipore)was carried out according to Ashida et al. (8).

Tissue Collection for RNA Isolation. Epidermal cells werecollected and pooled as described (11) from larvae on thesecond day of fifth instar at which developmental stagepro-phenol oxidase was found to be accumulating in body wallcuticle. Hemocytes were collected as described (12). Tissueswere stored in liquid N2 until used.RNA Extraction and Northern Blot Hybridization. Total

cellular RNA was prepared from pooled frozen epidermal cellsand hemocytes using a Stratagene RNA purification kit. Thepro-phenol oxidase hybridization probe consisted of a 32p_labeled random-primed 2.0-kb EcoRI fragment of hemocytepro-phenol oxidase (pPO17) cDNA (13). A 32P-labeled ran-dom-primed a-tubulin probe was used as a constitutively ex-pressed internal standard (14).

Assay of Phenol Oxidase Activity. Phenol oxidase activitywas assayed spectrophotometrically according to Ashida et al.(15). Under the assay conditions, activated homogeneouscuticular pro-phenol oxidase gives a specific activity (AA520 per5 min per mg of protein) of 3500-4000 (M.A., unpublisheddata).Assay of PPAE. PPAE activity was measured by the con-

version of its substrate, pro-phenol oxidase, to phenol oxidaseand is expressed in terms of the increase of phenol oxidaseactivity over time.

Protein Quantification. The protein concentration was mea-sured by using the Micro BCA protein assay reagent (Pierce)using bovine serum albumin as a standard.

RESULTSAppearance of Phenol Oxidase Activity in the Incubation

Mixture Consisting of the Pro-phenol Oxidase Fraction andthe AC Fraction in the Presence of Ca2 . Phenol oxidaseactivity increased with time when the pro-phenol oxidasefraction was incubated with the AC fraction that had beenpreviously treated with Ca2 . It was demonstrated that Ca2+was necessary for the AC fraction to acquire the activity toactivate pro-phenol oxidase in the mixture of both fractions(Fig. 1).

Presence of Pro-phenol Oxidase in the Pro-phenol OxidaseFraction. Anti-hemolymph pro-phenol oxidase IgG was mono-specific to hemolymph pro-phenol oxidase, which is composed

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FIG. 1. Activation of cuticular pro-phenol oxidase. The addition ofthe CM-ToyoPearl flow-through fraction containing pro-phenol oxi-dase to the Ca2+-treated AC fraction resulted in the appearance ofphenol oxidase activity (a). If the AC fraction was not treated withCa2+ and mixed with the pro-phenol oxidase fraction, no activityappeared (0). If, however, the AC fraction was mixed with thepro-phenol oxidase fraction and Ca2+ (final concentration 4 mM) wasadded at the same time, phenol oxidase activity gradually appeared(o). For all three experiments the mixtures were held on ice. Aliquotsof 30 ,ul of the mixtures were used to assay phenol oxidase activity,which is expressed as AA520 per 30 Al per 5 min. (Inset) Appearanceof PPAE activity in the AC fraction during its incubation with orwithout 4mM CaCl2 at 25°C. Prior to mixing the AC fraction with thepro-phenol oxidase fraction (1:1, vol/vol), the AC fraction was incu-bated at 25°C in the presence or the absence of 4 mM CaC12 for up to30 min. At the indicated times, the AC fractions were added to thepro-phenol oxidase fractions and incubated on ice for 5 min afterwhich phenol oxidase activity (30 ,ul of the mixture) was assayed. Theobserved phenol oxidase activity (AA520 per 30 Al per 5 min) corre-sponded to the PPAE activity generated during incubation with Ca2+at 25°C. 0, PPAE activity in the AC fraction incubated in the presenceof Ca2+; o, PPAE activity in the AC fraction incubated in the absenceof Ca2+.

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of two polypeptides with mobilities in SDS/PAGE corre-sponding to 71 kDa and 70 kDa (16). This antibody gaveidentical results with regard to cross-reactive polypeptides inthe cuticular extract (i.e., 71 and 70 kDa; Fig. 2, lane c) but didnot detect any polypeptides in the AC fraction, indicating thepresence of pro-phenol oxidase only in the pro-phenol oxidasefraction. It was confirmed by using homogeneous hemolymphpro-phenol oxidase that components other than pro-phenoloxidase in the cuticular pro-phenol oxidase fraction are notinvolved in the appearance of phenol oxidase activity (data notshown).

Factor(s) Responsible for Pro-phenol Oxidase Activation inthe AC Fraction. One of the factors responsible for pro-phenoloxidase activation in the AC fraction was shown to be a specificprotease because pro-phenol oxidase was converted to asmaller polypeptide doublet in SDS/PAGE during incubationof pro-phenol oxidase fraction with Ca2+-treated AC fraction.We will refer to the protease as pro-phenol oxidase activatingenzyme (PPAE). Furthermore, PPAE in the Ca2+-treated ACfraction was completely inactivated by DFP. On the contrary,DFP did not show any appreciable effect on the ability for theAC fraction to acquire PPAE activity when DFP had beenincubated with the AC fraction and subsequently removed bydialysis. These results strongly suggest that PPAE is a serineproteinase that exists as a zymogen (pro-PPAE) in the ACfraction. In the presence of Ca2+, the pro-PPAE is slowly

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FIG. 2. Detection of pro-phenol oxidase and phenol oxidase bySDS/PAGE and immunoblotting. (A) The SDS/PAGE pattern ofcuticular protein samples stained by Coomassie brilliant blue. Samplesapplied to each lane were as follows: lane a, marker proteins (phos-phorylase b, 92 kDa; bovine serum albumin, 66.2 kDa; ovalbumin, 45kDa; carbonic anhydrase, 31 kDa; trypsin inhibitor, 21.5 kDa; andlysozyme, 14.4 kDa); lane b, SDS/urea extract of cuticle; lane c,pro-phenol oxidase fraction; lane d,AC fraction; lane e, a mixture (1:1,vol/vol) of the pro-phenol oxidase fraction and the AC fractionincubated on ice for 30 min; lane f, mixture (1:1, vol/vol) of thepro-phenol oxidase fraction and the Ca2+-treated AC fraction incu-bated for 30 min on ice; lane g, marker proteins. The amount of proteinapplied to lanes c-f was 2.2 j,g, 1.3 j,g, 3.5 ,.g, and 3.5 j±g, respectively.To lane b, 2.5 ,ul of SDS/urea extract of cuticle was applied. (B)Immunoblotting of polypeptides cross-reactive to anti-(hemolymph)pro-phenol oxidase IgG after SDS/PAGE. Samples were identical tothose in A. The positions and sizes (in kDa) of marker proteins are

indicated at right.

activated in the mixture of pro-phenol oxidase fraction and ACfraction when incubated on ice (Fig. 1). However, at 25°C inthe presence of Ca2+ the pro-PPAE in the AC fraction israpidly converted to PPAE as shown by the rapid increase ofPPAE activity (Fig. 1 Inset).

It is not known at present whether components other thanpro-PPAE and Ca2+ are involved in the activation process ofpro-PPAE in the AC fraction.Immunoblotting with Anti-Hemolymph Pro-phenol Oxi-

dase IgG. Among proteins extracted from cuticles with 5%SDS, 3% 2-mercaptoethanol, and 5 M urea, no polypeptideother than a polypeptide doublet of pro-phenol oxidase cross-reacted with anti-hemolymph pro-phenol oxidase IgG (Fig. 2,lane b). The pro-phenol oxidase polypeptides were calculatedto be 71 kDa and 70 kDa, respectively, from their relativemobilities to the marker proteins. Native pro-phenol oxidaseappears to be a heterodimer of the polypeptides or a mixtureof two kinds of homodimers because the molecular mass ofnative pro-phenol oxidase was determined to be 128 kDa bygel-permeation chromatography (M.A., unpublished data).The mobilities of the pro-phenol oxidase polypeptides in thepro-phenol oxidase fraction were similar to those in theSDS/urea cuticular extract (Fig. 2, lane c). If the pro-phenoloxidase fraction is incubated with the AC fraction in theabsence of Ca2+, no difference in mobility could be detected(Fig. 2, lane e). If however, the pro-phenol oxidase fraction wasincubated with the Ca2+-treated AC fraction, a 4-kDa decreasein molecular mass of both polypeptides could be observed (Fig.2, lane f).Immunocytochemical Detection of Pro-phenol Oxidase in

Integuments and Trachea. Ultra-thin sections of integumentand trachea were stained with polyclonal rabbit anti-pro-phenol oxidase IgG (affinity purified) and subsequently witha goat anti-rabbit IgG-10-nm colloidal gold conjugate. Elec-tron micrographs of the stained sections are presented in Fig.3. Pro-phenol oxidase was localized in the nonlamellate en-docuticle, lamellate endocuticle, cuticular assembly zone, andalong the basal surface (hemocoel side) of the epidermalbasement membrane of the tracheal and body wall integument.Very few gold particles were detected in the cuticulin layer, inthe electron dense granules in the non-lamellate endocuticle,or in the underlying epidermal cells (Fig. 3 A and B). Aconspicuous feature of the labeling pattern in the lamellateendocuticle was that the gold particles were seen in an orderlyarray along the basal border of helicoidal chitin lamellae; otherportions of the lamellae were not labeled or very sparselylabeled (Fig. 3C). Contrary to this orderly labeling pattern inlamellate endocuticle, a random labeling pattern was observedin nonlamellate endocuticle (Fig. 3A). In trachea, the taenidialcushion was densely labeled for pro-phenol oxidase, but thedistribution pattern appeared random (Fig. 3D). Sparse label-ing of pro-phenol oxidase also was observed in the taenidialcuticle of the tracheae, whereas the endocuticle and cuticulinwere not labeled (Fig. 3D). Labeling in control sections stainedwith nonimmune rabbit IgG was insignificant (data notshown).Northern Blot Analysis of Pro-phenol Oxidase mRNA in

Epidermal Cells. As shown in Fig. 4, abundant pro-phenoloxidase mRNA was detected in total hemocyte RNA by a32P-labeled random-primed EcoR I fragment of the pro-phenoloxidase cDNA probe. However, pro-phenol oxidase mRNAcould not be detected in total RNA extracted from epidermalcells. One might point out that the pro-phenol oxidase probeused here was produced from cDNA originating from hemo-cyte pro-phenol oxidase mRNA and thus argue that cuticularpro-phenol oxidase and hemolymph pro-phenol oxidase arenonhomologous. However, we have determined amino acidsequences of several peptide fragments obtained from peptidemapping of purified cuticular pro-phenol oxidase and foundthem to be identical to amino acid sequences deduced from the

Proc. Natl. Acad. Sci. USA 92 (1995)

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hemolymph phenol oxidase cDNA (M.A., T. Ozawa, M. Ochiai,and Y. Yasuhara, unpublished data). Thus, the present resultshows the absence of pro-phenol oxidase transcripts in theepidermal cells at the time when pro-phenol oxidase is beingaccumulated in cuticle. This leads us to postulate that hemolymphand cuticular pro-phenol oxidase are the same gene productsynthesized in the hemocytes, released into the hemolymph, andthen transported into cuticle.

DISCUSSIONOne of the distinguishing features of arthropods is theirexoskeleton, which provides them with a protective armoragainst the aggressions of the external environment. It hasbeen considered for the most part that the integument is onlyan inert physical barrier (1). Recently, however, we demon-strated that the integument can actively respond to minorinjury in the presence of microbial cell wall components(lipopolysaccharide and peptidoglycan) by de novo synthesis ofcecropin antibacterial peptides and relaying them to the zoneof cuticular aggression (11, 17).

Another common injury or defense response is cuticularmelanization. This response can be elicited by a mechanicalscratch or by microbial invasion. This defense response isknown to play a role in the sequestration of fungal pathogensas they attempt to penetrate the cuticle (18). It is well knownthat melanin is synthesized from phenolic substances by theaction of phenol oxidase (19). Lai-Fook (5) and Barrett (20)suggested that cuticular melanization resulted from injury bythe activation of a pro-phenol oxidase by a proteinaceousactivator. The presence of pro-phenol oxidase, however, hasnot been unambiguously demonstrated, and no informationabout its activation has been elucidated since. According toBarrett (6), the isolation and characterization of cuticularphenol oxidase as well as the relationships between hemo-lymph and cuticular phenol oxidases remain important unre-solved questions in cuticular biochemistry.Our present results clearly demonstrate that cuticular phe-

nol oxidase is truly in a zymogen form, which is activatedthrough a limited proteolysis by the serine proteinase PPAE,which is also in a zymogen form. Hence, two distinct steps havebeen elucidated in the activation of pro-phenol oxidase. Ca2+

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FIG. 4. Northern blot analysis of pro-phenol oxidase mRNA incuticular epidermal cells and hemocytes. Total RNA from pooledepidermal cells and hemocytes was analyzed by Northern hybridiza-tion. The blot was probed simultaneously with the 32P-labeled random-primed 2.0-kb EcoRI fragment of hemocyte pro-phenol oxidase(pPO17) cDNA and a tubulin cDNA. Lanes E and H received 17.9 ijgof epidermal cell total RNA and hemocyte total RNA, respectively. Tothe lanes labeled 1/10, 1/20, and 1/40, reduced amounts of hemocyteRNA were applied (1/1Oth, 1/20th, and 1/40th of the amount appliedto lane H, respectively). From separate experiments, pro-phenoloxidase mRNA and tubulin mRNA were proved to migrate to thepositions corresponding to 2.7 kb and 1.7 kb, respectively.

is necessary for the conversion of pro-PPAE to PPAE; how-ever, we do not know if Ca2+ is involved in this specific reactionor if it is involved in cascade events prior to this step. Themechanism by which injury or pathogen invasion set thecuticular cascade into motion remains unknown.

In addition, we were able to specifically localize the pro-phenol oxidase in both the body wall and tracheal cuticle. Wecontend that pro-phenol oxidase is being localized instead ofphenol oxidase, since integuments were dissected in highconcentrations (664 ,uM) of the trypsin-type serine proteinaseinhibitor APMSF. Immunolocalization of cuticular proteinsand peptides is common (21-24). However, to our knowledge,the conspicuous orderly arrayed localization pattern of pro-phenol oxidase we observed along the basal border of thelaminae in the lamellate endocuticle of the body wall isseemingly novel. The physiological significance of this patternremains unknown.With regard to the site of synthesis of cuticular pro-phenol

oxidase, our present results strongly suggest that the cuticularpro-phenol oxidase is originally synthesized in hemocytes. Theidentity of amino acid sequences between peptide fragmentsfrom cuticular pro-phenol oxidase and the deduced amino acidsequence of hemolymph pro-phenol oxidase cDNA corrobo-rated our contention and allowed us to use the hemocytepro-phenol oxidase cDNA as a molecular probe. Furthermore,we examined pro-phenol oxidase transcription at the timewhen pro-phenol oxidase is being accumulated in the cuticle,so if the epidermal cells were involved, we would have expectedboth pro-phenol oxidase mRNA and its gene product to bepresent in the epidermal cells. In previous studies (8, 12),pro-phenol oxidase was localized in oenocytoid and plasmato-cyte-type hemocytes. Other tissues examined by the incorpo-ration of [35S]methionine into anti-pro-phenol oxidase IgGimmunoprecipitates-i.e., fat body and epidermal cells-didnot synthesize detectable quantities of pro-phenol oxidase(12).Very recently, Sass and colleagues (22) showed not only that

cuticular peptides are synthesized by the epidermal cells butalso that they can also originate from hemocytes and betransported across the epidermis and enter into the cuticular

matrix. This could explain our observation of pro-phenoloxidase localization along the basal surface (hemocoel side) ofthe epidermal basement membrane of the tracheal and bodywall integument. The mechanism with which particular hemo-lyph protein is destined to be transported to cuticle remainsunknown.Our present results along with those of previous studies (8,

12) demonstrate the ubiquity of pro-phenol oxidase through-out the insect body (i.e., hemolymph and cuticle). The insecttissues are literally bathed in or surrounded by this enzymezymogen and its activating cascade. Defense functions havealready been attributed to the pro-phenol oxidase cascade, butwe are now questioning whether pro-phenol oxidase couldfulfill other functions, such as an oxygen transport like hemo-cyanin or hemoglobin because silkworm pro-phenol oxidasewas shown to be a protein homologous to arthropod hemocy-anin (13). One could speculate that pro-phenol oxidase picksup oxygen from both the vast body surface and tracheal systemand transports it through the hemolymph to the tissues.Further studies are necessary to validate or invalidate suchspeculation.

We sincerely thank Professor Fotis Kafatos for his encouragementand valuable discussions. We are grateful to Miss Yoshiko Koizumiand Mr. Takashi Ozawa (Institute of Low Temperature Science,Hokkaido University, Sapporo) and Won-Jae Lee (Institut Pasteur,Paris) for their technical support. This work was supported by researchand travel grants from the Japan Society for the Promotion of Science,Institut Pasteur, Japan Ministry of Education, Science and Culture(Grant 02454019), Japan Ministry of Agriculture, Forestry and Fish-eries (BMP 95-III-2-1-4), and La direction du Developpement et de laCooperation Scientifique Technique et Educative du Ministere desAffaires Etrangeres Francaises.

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Ratnakaran, R. & Binnington, K. (CSIRO, Melbourne), pp.195-212.

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& Brey, P. T. (1993) J. Invertebr. Pathol. 62, 1-8.19. Mason, H. S. (1955) Adv. Enzymol. 16, 105-184.20. Barrett, F. M. (1984) Arch. Insect. Biochem. Physiol. 1, 213-223.21. Wolfgang, W. J., Fristrom, D. & Fristrom, J.W. (1986)J. CellBiol.

102, 306-311.22. Sass, M., Kiss, A. & Locke, M. (1994) J. Insect Physiol. 40,

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Proc. Natl. Acad. Sci. USA 92 (1995)

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..; -i

u.,

:-.-'