protein trafficking in insect cuticle · 2001. 5. 3. · some cuticular proteins occur not only in...

INTRODUCTION

Years ago it was generally accepted and established in classictextbooks on comparative anatomy and entomology(Chapman, 1969; Weber and Weidner, 1974; Fretter andGraham, 1976; Meglitsch and Schram, 1991) that theintegumental cuticle of insects (1) protects the organism frommechanical and chemical injury; (2) serves as an externalskeleton; (3) is made up of components, i.e. proteins and chitinmolecules, that are synthesized by the epidermal cell layer; (4)is a non-living component of the insect organism and is notinvolved in metabolic processes; (5) as a non-living structureinhibits the growth of the insect body, and therefore has to berenewed in the course of ecdysis; (6) breaks down duringthe moulting process when all of its macromolecules aredecomposed to their monomers (amino acids, ormonosaccharides) by the enzymes of the exuvial fluid.

Recently, however, data have appeared in the literaturesuggesting that this picture is more complicated. Some proteinsof the ‘soft’ cuticle of Lepidopteran and Dipteran larvae aresynthesized not (Koeppe and Gilbert, 1973; Palli and Locke,1987; Sass et al., 1993) or not exclusively (Palli and Locke,1987; Wielgus, 1983; Wielgus et al., 1990) in the epidermis butin other tissues. For example larval storage proteins aresynthesized in fat body cells, secreted into the hemolymph andtransported to the cuticle in some Dipteran and Lepidopteraninsects (Koeppe and Gilbert, 1973; Schenkel and Scheller, 1986;Sass et al., 1991), while these proteins are secretedbidirectionally by the epidermal cells in other Lepidopteraninsect species (Palli and Locke, 1987; Sass et al., 1993). Proteins

taking part in the defense reactions of insects are produced bythe hemocytes, however some of them are present in the cuticle,too (Brey et al., 1993; Sass et al., 1993). It has also been clarifiedthat some proteins occur only in the larval cuticle while othersoccur only in the pupal or imaginal cuticle (Riddiford and Hice,1985; Wolfgang and Riddiford, 1986). Cuticular proteins mighthave characteristic localizations throughout the body/segmentparts (Cox and Willis, 1985; Wolfgang and Riddiford, 1986;Bouhin et al., 1992; Locke et al., 1994). It may be important thatsome cuticular proteins occur not only in the integumentalcuticle, but in the tracheal cuticle too (Sass et al., 1994).

The aim of our present work was to follow by immunoblot andimmunocytochemical methods the synthesis, secretion, transportand accumulation of several cuticle proteins in the LepidopteranManduca sexta during the last larval and pupal stages. Our resultsshow that one group of cuticle proteins is synthesized in theepidermis and secreted apically into the larval cuticle anddecomposed during the moulting process. A second group ofproteins of the integumental cuticle are synthesized in the fat bodyand transported into the integument. Other proteins are transportedfrom the cuticle back to the hemolymph and stored in the fat bodybefore ecdysis, or reappear in the pupal cuticle again. This meansthat the cuticle is a living component of the organism and itsmaterials may be involved intimately and actively in delicatelyregulated processes of the postembryonal development.

MATERIALS AND METHODS

Experimental animalsTobacco hornworm (Manduca sexta) eggs were kindly provided by

2113Journal of Cell Science 112, 2113-2124 (1999)Printed in Great Britain © The Company of Biologists Limited 1999JCS0227

In the course of this study more than 20 proteins have beenisolated from the larval cuticle of Manduca sexta. Synthesis,secretion, transport and accumulation of four particularproteins, representative members of four characteristicgroups, were followed during metamorphosis byimmunoblot and immuncytochemical methods and aredescribed in detail in this paper. We established that onlysome of the proteins of the soft cuticle of Lepidopteran larvaeare synthesized in epidermal cells at the beginning of thelarval stages and are digested during the moulting period(MsCP29). Other proteins (MsCP30/11) are secreted into thecuticle by the epidermal cells in different forms duringvarious developmental stages. Some proteins are secretedapically during the feeding period, but before ecdysis they

are then taken up by epidermal cells and transported in abasolateral direction back into the hemolymph and saved inan immunologically intact form by the fat body cells(MsCP12.3). Some cuticle proteins have a non-epidermalorigin. They are transported from the hemolymph into thecuticle. Before and during ecdysis these molecules reappearin the hemolymph and are detectable again in the pupalcuticle (MsCP78). Our data prove that the cuticle is not anon-living part of the insect body: it is not only an inert,protective armor, but maintains a continuous and dynamicmetabolic connection with the other organs of the organism.

Key words: Insect cuticle protein, Secretion, Protein trafficking,Metamorphosis

SUMMARY

Insect cuticle, an in vivo model of protein trafficking

György Csikós, Kinga Molnár, Noémi H. Borhegyi, Gábor Cs. Talián and Miklós Sass

Department of General Zoology, Eötvös Loránd University, Budapest, 1088 Puskin u. 3, Hungary*Author for correspondence (e-mail: [email protected])

Accepted 13 April; published on WWW 10 June 1999

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Prof. S. E. Reynolds (University of Bath). Larvae were rearedaccording to his instructions based on the techniques of Bell andJoachim (1976), on a wheat germ based artificial diet, at 25°C,17L:7D photoperiod and 60% relative humidity. Animals wereselected that ecdysed to the fifth instar between 22.00 and 02.00 AZT(Arbitrary Zeitgeber Time). They began feeding 3 hours after ecdysison day 0. Growth was completed on day 4 subsequent to which theyinitiated wandering behaviour. Different stages of development wererecognised by a staging scheme adapted from Samuels and Reynolds(1993). When newly ecdysed pupae were required, larvae that hadceased feeding and initiated burrowing behaviour were allowed toundergo metamorphosis in glass tubes so the exact time of larval-pupal ecdysis could be ascertained.

Sample preparationHemolymphSamples were collected by puncturing the prolegs and drops ofhaemolymph were allowed to drip onto crystals of phenylthiourea inan Eppendorf tube. Samples were centrifuged for 5 minutes at 3000 gto pellet the hemocytes. The supernatant was diluted to twice itsvolume with homogenisation buffer which contained 100 mM sucrose,1% dextran, 40 mM Tris maleate (pH 7.2), 0.5 mM MgCl2, 100 mMKCl, 10 mM NaCl, 5.0 mM β-mercaptoethanol and 10 mM PMSF.

EpidermisThe last segment of the caterpillar was cut off and the carcass wasplaced on a glass surface between parallel strips of sticky tape. A testtube rolled from head to tail caused internal organs to be extrudedfrom the cut end. The integument skin was washed and the epidermissqueezed out by rolling with more pressure and no clearance tapes.The epidermal cell layer was collected in 100 µl of homogenizationbuffer. Samples were observed under a stereomicroscope for possibleundesired contamination with cuticle or muscles.

CuticleThe integumental sheet was rolled out until stereomicroscopeobservation showed it no longer contained epidermis. Pure cuticle wascut into small pieces and collected in an Eppendorf tube containing200 µl of homogenization buffer.

Internal organsFat body lobes, salivary glands, midgut, gonads and tracheae wereremoved from dissected caterpillars as quickly as possible. They wererinsed in a large volume of ice-cold homogenization buffer and allcontaminating tissues were very carefully removed under astereomicroscope. Tissue samples were quickly blotted and collectedin 50, or 100 µl of homogenization buffer.

Samples were homogenized in a glass-Teflon homogenizer and thedebris was removed by a short centrifugation. Freshly preparedsamples were boiled for 3 minutes with an equal volume of SDSsample buffer (Laemmli, 1970).

Analytical gel-electrophoresisSDS-PAGE was carried out according to the method of Laemmli(1970) on a 7.5% separating gel overlaid with a 3% stacking gel in aBio-Rad Minigel chamber. Gels were usually stained with CoomassieBBR to visualize the peptides. The molecular mass of the peptideswas determined by coelectrophoresis with standards from the Bio-Radlow (or sometimes the broad) molecular mass calibration kit.

Protein purificationCuticle peptides were separated on a 12%, or 10% polyacrylamidegel, respectively, in a Bio-Rad Prep Cell (Model 491) chamber. Gelswere run overnight at 10°C, 100 mA, and the peptides eluted from thegel column at 120 mA. The volume of collected fractions was 1.5 ml.Fractions were dialysed overnight against distilled water at 4°C and

concentrated in a SpeedVac system (RC10-10, Jouan). Purity ofindividual fractions was tested by analytical gel electrophoresis andthe fractions containing the same peptides in an electrophoreticallypure form were pooled and stored at −70°C until use.

Antibody developmentC57/Black mice were immunised with 25-30 µg of isolated andelectrophoretically pure peptides, dissolved in 100 µl of PBS andemulsified with 100 µl of Freund’s complete adjuvant. Animals wereboosted 3 weeks later with the same amount of peptides dissolved in100 µl of PBS and mixed with 100 µl of Freund’s incompleteadjuvant. The titer of the antibodies was tested in blood samplescollected from the tip of the tail. Sometimes the animals had to beboosted three or four times. After completion of immunisation themice were bled and the IgG fraction was separated from the serum byammonium sulphate precipitation.

ImmunoblottingPeptides were electrophoretically transferred to a sheet of nitrocellulose(Bio-Rad, 0.2 µm pore size) according to the method of Towbin et al.,(1979) in a Bio-Rad MiniBlot chamber. Nonspecific binding sites ofnitrocellulose sheet were blocked by 5% Carnation non fat dry milkpowder in Tris buffer (0.15 M Tris-HCl, 0.5 M NaCl, pH 7.0). Afterwashing three times with TBS, sheets were incubated overnight at 4°Cin the presence of first antibody diluted, usually 1:1000, in TBS. Blotswere washed again with TBS and incubated in the presence of secondantibody (AP-conjugated anti-mouse antibody developed in rabbit;Sigma Immuno-Chemicals) for 1 hour at room temperature. After finalwashes in TBS and TTBS (TBS containing 0.5% Tween-20) blots weredeveloped using freshly prepared NBT-BCIP (5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium, Sigma) as substrate. Molecularmasses were determined using the low range (and sometimes by thebroad range) prestained standards of Bio-Rad.

ImmunohistochemistryAnimals were fixed in 2% paraformaldehyde in 0.1 M cacodylatebuffer for 4 hours at 4°C, or in Bouin fixative for 6 hours at roomtemperature. Fixatives were injected into the body cavity then theanimals immersed into a large volume of it. After 2 hours the partlyfixed body was cut into three pieces and fixed further. Fixed materialswere embedded in Paraplast (Dulbecco). 5 µm serial sections were cut,placed on poly-L-lysine-coated slides and dried overnight at 39°C.

After rehydration and blocking (5% Carnation non fat dry milkpowder in PBS) sections were incubated overnight at 4°C in thefirst antibody, diluted 1:50. After washing alkaline-phosphatase-conjugated rabbit anti-mouse antibody (Sigma Immunochemicals)was applied at a 1:100 dilution for 1 hour at room temperature.NAMP-Fast-Red tablet sets (Sigma Fast, Sigma Chemical Co.) wereused for development. Some sections were incubated in the presenceof non-immune mouse serum. These sections remained absolutelynegative in each case.

Slides were mounted in Mowiol, and photographs were taken onKodak Gold 100 ASA film with a Zeiss Axioskop (Germany, MC80.Microscope Camera, OPTON) using DIC (differential interferencecontrast) optics.

As a result of the aldehyde fixation a pale green autofluorescenceemission was observed using violet blue excitation on the non labelledcells and tissue components (Del Castillo et al., 1989), while the FastRed positive structures were brillant purple under these conditions. Wefound that fluorescence microscopy gave a much better picture andmuch higher resolution to analyse the Fast Red developed sections thanthe common red-and-white pictures using simple visible illumination.

ImmunocytochemistryFor immunogold labelling samples were prepared according to themethod of Locke (1994) to preserve the antigenicity of the proteins inultrathin sections. Immunogold labelling was performed by following

G. Csikós and others

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a procedure simplified from that of Leung et al. (1989). Sections wereviewed in a JEOL JEM100 CXII electron microscope. The specificdifferences in labelling patterns obtained from each antibody served ascontrols in addition to those mentioned by Leung et al. (1989).

mRNA isolationIntegumental sheets were quickly prepared and frozen immediately inliquid nitrogen. The pooled samples were homogenised in ice-coldguanidine HCl and RNAs were isolated according to the method ofBerger (1987). The total cellular RNA was chromatographed on anoligo(dT) cellulose column to enrich the polyadenylated RNA fraction(Maniatis et al., 1987).

In vitro translationRabbit reticulocyte lysate cell free translation system (Promega) wasprimed with graded quantities of isolated mRNAs according to thepackaged protocol. After cell free translation for 1 hour at 37°C,translation products were electrophoresed and transblotted onto anitrocellulose sheet. Using the appropriate antibodies the presence ofthe peptide of interest in the translation product was investigated onwestern blots.

Hormone treatments20-hydroxyecdysone (Sigma) and ecdysteroid agonist RH-5849(kindly given by Prof. S. E. Reynolds) were injected (10 µg) in 10 µlof ethanol into the dorsal part of the thoracal segments using aHamilton syringe with fixed 28 swg needle. Control animals weregiven 10 µl of ethanol alone.

HistoblotsPieces of integument, 5-7 mm2 in size, were cut from the lateralabdominal segments and the epidermal part was detached carefullyfrom the cuticle using very fine, sharpened forceps. The preparationswere rinsed in Ringer solution and placed between two nitrocellulosesheets soaked with Grace’s medium. After incubation for 90 minutesin a wet chamber, at room temperature the membranes were handledand developed as for the immunoblots.

RESULTS

Isolation of cuticular proteins and their distributionbetween various organs of the caterpillar detectedby western blotsCuticle proteins of the larvae on the 4th day of the last larvalstage were electroeluted on 12%, or 10% polyacrylamide gels,respectively, in a preparative chamber. A 12.3 kDa protein(MsCP12.3) was present in an electrophoretically pure form infractions 135-144, a 29 kDa protein (MsCP29) in fractions291-316 and a 30 kDa protein (MsCP30) in fractions 324-343.Other proteins were isolated from a 10% gel and fractions 440-455 contained a 78 kDa protein (MsCP78) (Fig. 1). Polyclonalantibodies developed against the MsCP12.3, MsCP29 andMsCP78 proteins reacted specifically with one, single band,with a molecular mass corresponding to that of the originallyisolated antigens on western blots of total homogenate of larvalcuticle (Figs 2a, 3a and 5a). Antibody developed against theMsCP30 protein labelled the corresponding 30 kDa band andalso a 11 kDa band on cuticle samples (Fig. 4a).

The antibody developed against MsCP12.3 recognised theoriginally isolated protein on western blots in integumentalepidermis on days 1-4 and in cuticle on days 3-8 of the last larvalstage. Until the prepupal period this protein was not detectablein any other tissues (tracheae, hemolymph, fat body, midgut,

salivary glands, gonads) of the larval organism (Fig. 2a). In theprepupal period this protein appeared in the hemolymph andduring the moulting process it was accumulated in the fat body(Fig. 2b). In freshly moulted (green) pupae when the formationof pupal endocuticle occurs it appeared mainly in the epidermisbut also in the cuticle. On the 2nd day of the pupal stage it waspresent in the epidermis but its concentration became muchhigher in the cuticle (Fig. 2c).

MsCP29 was continously present in integumental andtracheal epidermis and cuticle from the 2nd day of the lastlarval stage until the beginning of the larval-pupal moult onwestern blots (Fig. 3a). During the wandering period itdisappeared from the epidermal cell layer (Fig. 3b).

The behaviour of MsCP30 was different from the abovementioned two proteins. After the electrophoretic isolation itoccurred in pure form in fractions 324-343 and no otherproteins were detectable in these fractions. A polyclonalantibody developed against this particular protein recognisedanother protein in cuticle samples prepared on the 5th day ofthe last larval stage. The molecular mass of this second positivepeptide was 11 kDa. The original antigen (MsCP30) waspresent in the epidermal cell layer during the last larval stageand on the first two days of the pupal stage, while the 11 kDaprotein occurred in epidermis until the beginning of thewandering period (Fig. 4a). This antibody gave only a positivereaction in integumental cuticle with the 11 kDa protein duringthe feeding period. Both of the positive bands were observablein the course of the wandering stage. Interestingly, in pupalcuticle samples only the MsCP30 band reacted with theantibody against MsCP30/11 (Fig. 4b).

The fate of MsCP78 was different again. According to thewestern blots it was present in the epidermis during the feedingperiod. It was also observed in fat body samples on the 1st and2nd days and in the hemolymph on the 2nd day of the last larvalstage (Fig. 5a). It was detectable in cuticle samples from the 3rdday of the larval stage until the beginning of apolysis. In thecourse of apolysis this protein reappeared again in hemolymphsamples (Fig. 5b). In 48-hour-old pupae the MsCP78 wasdetectable only in the epidermis and cuticle (Fig. 5c).

Fig. 1. Electrophoretogram of the four peptides isolated from thecuticle of Manduca sexta larvae. (Coomassie BBR stain) Lane C:protein pattern of cuticle homogenate, lanes labelled as 12.3, 29, 30and 78 show the bands of isolated proteins.

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Immunohistochemical resultsDuring the feeding period MsCP12.3 was observable at thelight microscope level exclusively in the epidermal cell layerand in the cuticle. It gave a strong, positive reaction in thecytoplasm of the epidermal cells, while all of the other tissues

remained negative on days 1-3 of the last larval stage (Fig. 6a).From day 3 of the last larval stage it was present in the exo-and endocuticlar layers of the integumental cuticle (Fig. 6b),but not in the tracheal cuticle. During apolysis MsCP12.3 wasclearly observable in the moulting fluid (Fig. 6c). In the course


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Fig. 2. (a) Evidence for the presence of MsCP12.3 in thehomogenates of various tissues of Manduca larvae on the second dayof the last larval instar. The protein of interest could be detected onlyin the epidermis (Ep) and cuticle (C); all of the other tissues werenegative. (b) Immunoblot of samples prepared from the tissues oflarvae at the time of apolysis showed a redistribution in thelocalization of the MsCP12.3. While the protein purified from thelarval cuticle disappears from the cuticle (C), it appears in thehemocyte-free hemolymph (H) and fat body (Fb). Samples preparedfrom the other tissues were invariably negative. (c) Antisera againstMsCP12.3 recognized the protein in the homogenate of pupalepidermis (Ep) and cuticle (C) of freshly moulted (P0) and two-day-old (P2) pupae. epidermis Ep; cuticle C; trachea, Tr; hemolymph, H;fat body, Fb; midgut, Mg; salivary gland, Sg; gonad, G; Bio-Radbroad range prestained molecular mass standard, St.

Fig. 3. (a) Immunological evidence for the presence of MsCP29 inhomogenates of epidermis (Ep), cuticle (C) and trachea (Tr) on thesecond day of the last Manduca larval stage. Samples of the othertissues did not show any positive reaction. (b) At the end of the lastlarval instar MsCP29 cuticular protein was present exclusively in thesamples of the cuticle (C) and tracheae (Tr), while the other tissuesremained negative. For abbrevations see Fig. 2 legend.

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of the moulting process it was accumulated in the trophocytesof the visceral lobes of the fat body (Fig. 6d).

At the electron microscope level MsCP12.3 showed acontinuous distribution in the lamellar cuticle from theassembly zone to the epicuticular layer (Fig. 7a).

MsCP29 was present only during the larval period ofpostembryonal development. It occurred in the epidermal celllayer of the integument (Fig. 8a) and of the tracheal system tillthe beginning of the wandering period. On the 2nd day of thelast larval stage it appeared in integumental (Fig. 8b) and

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Fig. 4. (a) Antiserum raised against MsCP30 recognised two bandson western blots with molecular masses of 30 kDa and 11 kDa.These proteins showing immunological identity appeared only in thesamples of the integumental epidermis and/or cuticle and also in thehomogenate of the tracheae. On the first day of the last larval stage(D1) both of the proteins were present in the epidermis (Ep) but onlythe smaller one was detectable in the cuticle (C). During the feedingperiod (D2-D4) the immunoreaction of MsCP30 was strong. At thesame time only the 11 kDa protein was detectable in the cuticle.(b) Immunoblot developed by the antiserum against MsCP30demonstrates that the 11 kDa protein was not present in theepidermal samples from the beginning of the wandering period (D5-D6) till the larval/pupal moult (D7). At the same time the cuticlecontained continously the 11 kDa protein and the 30 kDa form alsoappeared. Interestingly, in pupal epidermis and cuticle (P2) only theMsCP30 was detectable on the immunoblots. For abbrevations seeFig. 2 legend.

Fig. 5. (a) Immunological evidence for the presence of MsCP78 inthe samples of epidermis (Ep), hemolymph, (H) and fat body (Fb) ofthe two-day-old Manduca sexta larvae. (b) At the end of the lastlarval instar MsCP78 was present exclusively in the samples ofcuticle (C) and hemocyte-free hemolymph (H) according to theresults of immunoblots. (c) After larval/pupal moult MsCP78reappears in the epidermis and cuticle of the freshly moulted (P0)and two-day-old (P2) pupae. For abbrevations see Fig. 2 legend.

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tracheal (Fig. 8c) cuticle and then disappeared from thesestructures during the larval-pupal moult (Fig. 8d). This proteincould not be detected by immunohistochemistry in any othertissues of the caterpillar.

Localization of MsCP30 was quite similar to that ofMsCP29 during larval development. It was present in theintegument (Fig. 9a) and in the tracheal system (Fig. 9b) fromthe first day of the last larval stage and disappeared during themoulting period. The antibody gave a strong, positive reactionin cuticular layers except in the assembly zone just above theepidermal cells. As apolysis began this protein seemed to havebecome decomposed since it was not observed in the exuvialspace. Only a remnant of the old cuticle containsimmunopositive material on histological preparations (Fig. 9c).It was detected in the pupal cuticle on sections (Fig. 9d)although it gave a very weak positive reaction on western blots.

Immunogold localization shows that the antiserumdeveloped against MsCP30 reacted exclusively with thelamellate zone of the integumental cuticle (Fig. 7b). In thetracheal system MsCP30/11 had a characteristic localizationwhich appeared mainly in the upper part of the endocuticle oftaenidia both of the tracheae and tracheolae (Fig. 7c).

At the light microscope level MsCP78 was found in thevisceral lobes of the fat body on the 1st and 2nd days of the lastlarval stage (Fig. 10a) and some hours later it appeared in theintegumental epidermis (Fig. 10b). From the 3rd day it appearedin the integumental cuticle (Fig. 10c) both in the intra- andintersegmental areas. At the same time it disappeared from thefat body, probably because its synthesis and secretion had beenceased. During the wandering period the MsCP78 disappearedfrom the epidermal cell layer. At the prepupal period, beforeapolysis, it was detectable in the hemolymph (Fig. 10d). Thisprotein was present in the pupal epidermis and cuticle.

In vitro translationmRNAs were prepared from the epidermis, fat body, midgut

cells and hemocytes on the 1st and 2nd days of the last larvalstage. A rabbit reticulocyte in vitro translation system wasprimed by the isolated mRNAs. The translation product waselectrophoresed, transblotted onto nitrocellulose sheets anddeveloped using polyclonal antibodies against the isolatedproteins. MsCP12.3, MsCP29 and MsCP30 appearedexclusively in the in vitro translation product of epidermalsamples which means that their mRNAs were present only inthe epidermis. In this system the antibody developed againstMsCP30 did not recognise the 11 kDa band. MsCP78occurred not only in the samples prepared from the epidermalcells, but also in the fat body samples. The molecular mass ofthe immunopositive band from the in vitro translation productwas about 83 kDa, which was slightly larger than that of thecorresponding protein present in the homogenate of theepidermal cell layer (Fig. 11).

Determination of the vectorial secretion byhistoblotsOn histoblots of the epidermal cell layer of two-day-old larvaeMsCP12.3 was secreted in the apical direction and was notdetectable on the basolateral surface. In the prepupal stage thesituation was just the opposite; this protein did not give apositive reaction on the apical surface, but appeared on thebasolateral side of the preparation (Fig. 12).

On the first three days of the last larval stage MsCP29 wasdetectable on the apical surface of histoblots; later on the blotsdeveloped by this antibody were absolutely negative on bothsides.

Using antibody developed against MsCP30 the histoblotsgave the same results as for MsCP29. The presence of thisprotein was clearly seen on the apical surface of the epidermalcell layer during the first period of the last larval stage.

MsCP78 was secreted apically to the cuticle during thefeeding period. This process ceased on the 3rd day of the lastlarval stage. It was interesting that this particular protein


Fig. 6. Localization ofMsCP12.3 byimmunohistochemistry in theepidermal layer of theintegument after 12 hours of thelast larval-larval moult (A). Atthe beginning of the wanderingperiod the MsCP12.3 waspresent only in the procuticle ofintegument except the assemblyzone (B). During apolysisMsCP12.3 quickly disappearedfrom the cuticle while it waswell detected in the exuvialspace by immunohistochemicalmethods (C). At the time oflarval-pupal moult MsCP12.3was accumulated in thetrophocytes of the fat body lobes(D). Assembly zone, AZ; cuticle,C; epidermis, Ep; epicuticle,Ec, exuvial space, ES; fat body,Fb; hemolymph, H; midgut, Mg;muscle, M; salivary gland, Sg;trachea, Tr. Bars: 25 µm (a);50 µm (b,c); 100 µm (d).

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appeared on the basolateral surface of the epidermis inprepupae, just before apolysis, as did MsCP12.3.

Effect of 20-hydroxyecdysone treatment onsynthesis and secretion of isolated proteinsLarvae were injected with 20-hydroxyecdysone 2-3 hours afterthe last larval-larval moult. 8 hours later a drastic decrease inthe amount of the MsCP12.3 (Fig. 13a) and MsCP29 (Fig. 13b)proteins was observed in the epidermis of treated animals. 8hours after the injection of exogenous 20-hydroxyecdysone theMsCP30 disappeared from the epidermal cells and only tracesof 11 kDa protein were present in the same samples (Fig. 13c).

In the second experiment the larvae were treated with 20hydroxyecdysone 24 hours after the last larval larval moult. 8hours after the injection the amount of MsCP12.3 and MsCP29decreased in epidermal samples and MsCP30 completelydisappeared, while the immunopositive 11 kDa band showed astronger reaction on western blots (Fig. 13a,b,c).

In the third experiment 24-hour-old larvae were injectedwith a moulting hormone analogue, RH5849 instead of 20-hydroxyecdysone. Due to the effect of this administration theconcentration of MsCP12.3, MsCP29 and MsCP30/11decreased in epidermal cells and all of the three proteins wereundetectable in the samples.

Fig. 7. (a) Immunogoldlocalization of MsCP12.3 on anultrathin section ofintegumental cuticle on thesecond day of the last larvalstage shows that the proteinwas present in the lamellatedendocuticle. Bar, 0.3 µm. (b)According to the immunogoldlabelling the MsCP30/11protein was localized in thelamellated endocuticle too. Bar,0.5 µm. (c) The ultrastructurallocalization of the MsCP30/11protein proves that this proteinwas present only in theendocuticular structures of thetracheae (C) and tracheolae(insert). Bars: 1 µm; 0.5 µm(inset).

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20-hydroxyecdysone treatment was applied at the start of thewandering period too. 8 hours after the injection the presenceof MsCP12.3 was not detectable at all in the fat body lobes.

In the case of MsCP78 the 20-hydroxyecdysone andRH5849 treatments remained ineffective. The amount of thisprotein did not change in the epidermal or fat body cells underthe effect of the hormone and its analogue in each studieddevelopmental period (data not shown).

DISCUSSION

Insect cuticle is an extracellular structure secreted by theepidermal cell layer consisting of chitin filaments embedded ina protein matrix (Anderson et al., 1995). The composition andpattern of cuticular proteins may be categorised from variouspoints of view. Some of them are present in the cuticle in eachdevelopmental stage, but others are definitely stadium-specific


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Fig. 8. MsCP29 protein wasdetectable both in theepidermis and cuticle of theintegument on the first day ofthe last larval stage (A). At theend of the feeding period theMsCP29 was translocated andaccumulated into theintegumental (B) and tracheal(C) cuticle. The presence ofMsCP29 could not be detectedin the pupal integument (D)and tracheae byimmunohistochemicalmethods. For abbreviations seeFig. 6 legend. Bars: 50 µm(a-c); 25 µm (d).

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Fig. 9. MsCP30 was localizedonly in the integumental (A) andtracheal (B) cuticle after 12hours of the last larval-larvalmoult. During apolysisMsCP30/11 was continuouslypresent in the integumentalcuticle but it was not detectablein the exuvial space byimmunological methods (C).After the larval-pupal moultMsCP30/11 appeared again inthe procuticle of pupalintegument (D). Forabbreviations see Fig. 6 legend.Bars: 50 µm (a-c); 25 µm (d).


proteins (Snyder et al., 1982; Fechtel et al., 1989; Rebers andRiddiford, 1988; Horodyski and Riddiford, 1989; Apple andFristrom, 1991; Bouhin et al., 1992; Charles et al., 1992). Theymay be arranged on the basis of their specific localisation inthe body, or skeletal parts (Willis et al., 1981; Cox and Willis,1985; Kiely and Riddiford, 1985; Wolfgang and Riddiford,1986; Binger and Willis, 1994; Lampe and Willis, 1994) oraccording to their distribution in the various layers of thecuticule (Locke et al., 1994). Some of the cuticular proteinsoccur only in the integument, but others are also present in thetracheal cuticle (Sass et al., 1993). Proteins of the cuticle maybe sorted on the basis of their physiological functions. Besidethose that are responsible for sustaining skeletal functions,storage proteins and proteins with a role in defense reactions

also occur in the cuticle (Palli and Locke, 1986; Sass et al.,1993; Brey et al., 1993; Marmaras et al., 1996).

In the course of this study more than 20 proteins have beenisolated from the larval cuticle of Manduca sexta. Isolations werecarried out on the 4th day of the last larval stage, i.e. at a timebefore the reprogramming of the epidermal cells (Riddiford,1978). At this developmental period the mass of loosely boundcuticular proteins was large enough for them to be purified andtheir electrophoretic pattern was identical to the data publishedin the literature (Kiely and Riddiford, 1985; Wolfgang andRiddiford, 1986; Horodyski and Riddiford, 1989). Using specificantibodies we followed their fate by immunological methodsduring metamorphosis. Surprisingly, only one group of larvalcuticle proteins was synthesized exclusively by the epidermalcells, secreted apically into the cuticle and disappeared during the

a b

c d

Fig. 10. Antisera developedagainst the MsCP78 cuticularprotein gave a strong positivereaction in the visceral lobe ofthe fat body on the first day ofthe last larval-larval stage (A).MsCP78 appeared in epidermalcells on the second day of thelast larval stage (B). At thebeginning of apolysis MsCP78was localized in theintegumental cuticle (C)exclusively. At the time of thelarval-pupal moult the MsCP78was accumulated in thehemolymph (D). Forabbreviations see Fig. 6 legend.Bars: 100 µm (a); 50 µm (b,c);25 µm (d).

Fig. 11. The in vitro translation product primed by the RNAsprepared from the epidermal cells (Ep) and fat body (Fb) wastransblotted and the presence of the isolated proteins was provedusing the antibodies developed against them (blots labelled as 78,30/11, 29, 12,3).

Fig. 12. Histoblots developed by the antibody against MsCP12.3 onthe second day of the last larval stage show that it was secreted bythe epidermal cell layer only apically (A) and not basolaterally (B),but on the 7th day of the last larval stage it appeared on thebasolateral surface of the preparate (C).

A B C

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larval-pupal moult. All of the other studied proteins have adifferent origin, location, function, lifetime etc. (Table 1).

The fates of four particular proteins, representative membersof the four characteristic groups of cuticular proteins (labelled bybold letters in Table 1) are described in some detail in this study.

MsCP29 belongs to the group of ‘classic’ cuticular proteins(Kiely and Riddiford, 1985; Sass et al., 1993). It occurs solely inthe integumental and tracheal epidermis and in the integumental

and tracheal cuticle. According to histoblots it is secreted apicallyduring the first 1-3 days of the last larval stage and never appearson the basolateral surface of the integument. It gives a strong,positive, but diffuse immunoreaction in the exo- and endocuticle.In the course of the larval-pupal moult it disappears from the old,larval cuticle. Synthesis of this protein is negatively regulated by20-hydroxyecdysone similarly to most of the so far decribedcuticular proteins (Apple and Fristrom, 1991; Hiruma et al., 1991;Nakato et al., 1992). After long term (8 hours) hormone treatmentits amount is strongly decreased and after RH5847 treatment itwas eliminated from the larval epidermal cells.

MsCP30 belongs to another group of cuticle proteins,mainly because it occurs not only in larval, but in pupal cuticletoo. This protein occurs exclusively in the integumental andtracheal system. Its presence is detectable in the epidermisduring the last larval stage and the first two days of the pupalstage. It appears on the 5th day of the last larval stage in theexo- and endocuticular layers of the intra- and intersegmentalcuticle. Only the so called assembly zone is devoid of thisprotein. MsCP30 cannot be detected in the cuticle on westernblots during the larval-pupal moult. On the second day of thepupal stage it reappears in the pupal cuticle. According to theresults of histoblots, this protein is secreted only in the apicaldirection, never giving a positive reaction on the basolateralsurface of epidermal cells.

Western blots show that the antiserum developed againstMsCP30 also reacts with a smaller, 11 kDa protein. It might be


a

b

c

Fig. 13. (a) Western blot analysis of epidermal samples preparedfrom 20-OHE or an RH5849 treated larvae confirmed that thesynthesis of MsCP12.3 is negatively regulated by 20-OHE. Theadministration was applied after 2-3 hours to the last larval-larvalmoult. 8 hours later a strong decrease in the amount of MsCP12.3protein was observed in the homogenates of the epidermis of treatedanimals (E1) relative to the sample made from the Ringer-treatedlarvae (K1). If the larvae were treated 24 hours after the last larval-larval moult the results showed strong similarity to the previousobservations (E2 and K2). Equal amounts of proteins were loaded inthe lanes labelled E1 and K1 as well as E2 and K2. After RH5849treatment the MsCP12.3 completely disappeared from epidermalsamples (RH and K3). (b) Effect of 20-OHE or RH5849 treatment onthe synthesis of MsCP29 in epidermal cells of Manduca larvae.Moulting hormone treatment was applied 2-3 hours later to the lastlarval-larval moult and the epidermal samples were prepared 8 hoursafter the hormone injection. A significant decrease in the amount ofMsCP29 was observed in the epidermis of treated animals (E1)relative to the sample made from Ringer-treated larvae (K1). If thelarvae were treated 24 hours after the last larval-larval moult and thesamples from the epidermis were prepared 8 hours late, the observedresults were the same as in the previous experiments (E2 and K2).Using the RH5849 treatment the same, but stronger effect wasdetected (RH and K3). (c) Alteration of the pattern ofimmunopositive bands in epidermal samples after various treatmentsusing the antisera against the MsCP30. Larvae were treated by 20-OHE 2-3 hours after the last larval-larval moult. 8 hours later thepresence of MsCP30 could not be detected in epidermal samples oftreated animals (E1) relative to the sample prepared from Ringer-treated controls (K1). This treatment caused a decrease in the amountof 11 kDa protein (E1 and K1). The same experiment carried out on24-hour-old larvae gave a similar result but the immunopositive bandof the 11 kDa protein showed a stronger reaction (E2 and K2). UsingRH5849 instead of 20-OHE in the same experimental arrangementboth of the proteins disappeared from the epidermis (RH and K3).


due to the presence of a similar epitope on two different cuticularproteins, or to the alternative splicing of the same primary RNAtranscript. According to our interpretation a further possibleexplanation may be that the 11 kDa protein originates fromMsCP30 after a posttranslational modification, i.e. after acleavage. This way the 11 kDa protein is an identical part of theMsCP30 and our antibody recognises the same epitope(s) on it.This supposed cleavage might occur in epidermal cells duringthe feeding period before the protein is secreted into the cuticle.During this period of postembryonal development the 30 kDaform of the protein is present in the epidermal cells, but only the11 kDa form of it is secreted into and accumulates in the cuticle.At the beginning of the wandering stage there is a switch; fromthis point of time the 30 kDa form of MsCP30 appears on theblots of cuticle samples too indicating that its secretion into thecuticle begins. On the basis of these results one can speculatethat the enzyme responsible for the supposed cleavage is notsynthesized or does not work after the wandering period. Thesynthesis of this hypothetical enzyme may be downregulated bythe commitment peak of 20-hydroxyecdysone which induces thewandering period itself.

Both the MsCP30 and the 11 kDa proteins are clearlyobservable on Coomassie BBR stained gels and fluorograms ofManduca cuticle proteins published earlier (Kiely and Riddiford,1985; Wolfgang and Riddiford, 1986; Horodyski and Riddiford,1989). Although their synthesis and fate were not analysed indetail, it is clear from the figures that the 11 kDa protein ispresent in the epidermal cells only during the feeding period,while the synthesis of MsCP30 is continously observable on thegels in the samples prepared from the epidermis of Manducasexta even to the end of the pupal stage (Kiely and Riddiford,1985), which is in good agreement with our results.

MsCP12.3 represents another very interesting and so farunknown group of cuticular proteins. It can be localised byimmunological methods in epidermal cells on days 1-4 of thelast larval stage and in the cuticle from the 3rd day till thebeginning of apolysis of the larval-pupal moult. mRNAsencoding this protein occur only in the epidermal cells and areabsent from all other investigated tissues. During the feedingperiod it is secreted apically as the histoblots show clearly.Surprisingly, in the prepupal period the MsCP12.3 is detectableon the basolateral and not on the apical surface of histoblots. Atthe same time this particular protein appears in the hemolymph,and the amount in the cuticular sheet quickly decreases. Beforepupation it accumulates in fat body cells. During ecdysis the

antibody gives a very strong, positive reaction exclusively introphocytes. These results suggest that during the prepupalperiod MsCP12.3 is taken up from the cuticle by epidermalcells, transported basolaterally to the hemolymph andendocytosed by the trophocytes in an immunologically intactform. It could indicate that MsCP12.3 may have some kind ofphysiological role later during metamorphic processes.

Although synthesis, transport and accumulation ofMsCP12.3 was not analysed in detail by other investigators aprotein with the corresponding molecular mass is clearlyvisible on gels and fluorograms published earlier (Kiely andRiddiford, 1985; Wolfgang and Riddiford, 1986; Hordyski andRiddiford, 1989). It was also shown that MsCP12.3 issynthesized exclusively during the feeding period of the lastlarval stage (Kiely and Riddiford, 1985; Wolfgang andRiddiford, 1986) which corresponds well with our results.

MsCP78 is a member of a fourth group of cuticle proteins.It appears in the epidermal cell layer and in the cuticle 2-3hours after the L4-L5 moult, but interestingly, it is present ina very large amount in the fat body cells too. On the secondday of the last larval stage MsCP78 is detectable in thehemolymph, but from the 3rd day it could be detected only inthe cuticle. In prepupae it is detectable again in thehemolymph. Histoblots clearly verify this observation. Theygive a positive reaction on the apical surface of the epidermallayer during the feeding period, but in the prepupal stagehistoblots are positive only on the basolateral surface.According to the results of in vitro translations this particularprotein is synthesized in the fat body and in epidermal cells ina larger (83-85 kDa) form. Our observations suggest that aconsiderable amount of MsCP78 is produced by the fat body,secreted to the hemolymph and enters into the cuticle from thehemolymph by transepithelial transport. This process isreversed at the prepupal stage, when this particular protein istransported basolaterally, from the cuticle to the hemolymph.

A protein with a similar molecular mass has been isolatedfrom the fat body lobes of Manduca sexta by Wielgus andGilbert (1978). Based on 3H leucine incorporation studies thisprotein was identified as hemolymph trophic factor (HTF). Themolecular mass of the larger subunit of HTF is about 75 kDa.It is synthesized in the fat body during the feeding period,secreted to the hemolymph and transported to the cuticle sincethe labelled protein is extractable from the larval cuticle(Wielgus, 1983; Wielgus et al., 1990). The further fate of theHTF was not analysed in these earlier studies.

To sum up, our results strengthen the view that the softcuticle of Lepidopteran larvae is not a non-living part of theorganism and not all of its components are digested during themoulting periods as happens with the best known cuticleproteins (MsCP29). Some of the proteins (MsCP30/11) aresecreted into the cuticle by the epidermal cells in differentforms in different developmental stages. Others, secretedapically during the feeding period, are then taken up byepidermal cells and transported in a basolateral direction backinto the hemolymph and saved in an immunologically intactform by the fat body cells (MsCP12.3). Some other cuticleproteins are transported from the cuticle into the hemolymphbefore ecdysis, but after the larval-pupal moult these proteinsare detectable again in the cuticle (MsCP78).

The data show that the cuticle is not an inert, protectivearmor, but that it maintains a continous and dynamic metabolic

Table 1. Classification of isolated cuticle proteinsMsCPs synthesized

MsCPs synthesized not exclusively by MsCP secreted by the epidermis epidermis and apicaly but later and secreted transported to MsCPs modified on basolateralyapicaly the cuticle after translation by the epidermis

MsCP24 MsCP55 MsCP30/11 MsCP12.3MsCP26 MsCP60 MsCP41/34MsCP29 MsCP65 MsCP68/40MsCP38 MsCP71MsCP40 MsCP78MsCP58 MsCP84MsCP77 MsCP110MsCP80MsCP86MsCP95

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connection with the other organs of the organism. Therefore,larvae do not have to decompose and resynthesize in eachmoulting period all of the proteins of their exoskeletal/trachealsystems, which represent a considerable amount of material.

Further studies are in progress to disclose the functions ofthe saved proteins during the metamorphic processes and toelucidate the molecular details of sorting/targeting signalsnecessary to the observed and described protein trafficking.

This work has been supported by the National Scientific ResearchFund (OTKA) under grant No. T014758, provided to M.S..

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