Thaumetopoein: An urticating protein from the hairs and integument of the pine processionary caterpillar (Thaumetopoea pityocampa schiff., Lepidoptera, Thaumetopoeidae)

Taxiton, Vol . 24, No. ", pp. 347-356, 1986 . Printed In Great Brhaia .

0041-0101/86 x3.00+ .00 C 1986 Perpmon Press ltd .

THAUMETOPOEIN: AN URTICATING PROTEIN FROM THE HAIRS AND INTEGUMENT OF THE PINE PROCESSIONARY CATERPILLAR (THAUMETOPOEA PITYOCAMPA SCHIFF ., LEPIDOPTERA, THAUMETOPOEIDAE) MICHEL LAMY,' MARIE-HÉLÈNE PASTUREAUD,' FRANÇOISE NOVAK,' GEORGES DUCOMBS,~ PHILIPPE VINCENDEAU, = JEAN MALEVILLE 3 and LUCIEN TEXIER = `UniversitE de Bordeaux I, Laboratoire de Neuro-Endocrinologie, U .A . 683 du C .N .R.S ., Avcnue des Facultés, 33405-Talen«-Cedex, and, 'Clinique dermatologique, Hbpital Saint André, 1 rue Jean Burguet 33073-Bordeaux-Cedex, France

(Accepted forpubJirntion 30 September 1985) M . LM+v, M .-H . P~stvxF.~Un, F . Novwx, G . DucoMas, P. VllvceNnewu, J . MALEVILLE and L . Tt:xtEq. Thaumetopoein : an urticating protein from the hairs and integument of the pine proceaaionary caterpillar (Thaumetopoea pityocampa Schiff ., Lepidoptera, Thaumetopoeidae) . Toxicon ?A, 347 - 356, 1986 . - Hairs of the Thaumetopoea pityotampa caterpillar (Lepidoptera) cause a cutaneous reaction in man and animals. The irritating fraction extracted from hairs rnntaina soluble proteins which were separated by various electrophoretic and immunoelectrophoretic techniques. Some of these proteins are present also in cuticle and haemolymph . One protein of 28,000 mol . wt is hair specific and caused a reaction in pig akin identical to that produced by hair extract . It is therefore an urticating protein which we have named thaumetopoein . This protein is formed of two subunits of molecular weights 13,000 and 15,000. It is present in large quantities in the glands producing urticating hairs .

INTRODUCTION HAIRS of the pine processionary caterpillar (Thaumetopoea pityocampa Schiff .) cause a contact dermatitis in man known as erucism (LAMP, 1977) . Following DEMOLIN'S (1%3) initial light microscope morphological investigation, the urticant apparatus was later studied using a scanning electron microscope (Bergsud, Doctorat Thesis, University of Bordeaux, 1979; DUCOMBS et al., 1979). Urticating hairs formed in caterpillars are not present in moths, which possess only scales which vary in size and form . Hairs are unable to regenerate after experimental depilation (LAMP et al., 1982x). The hairs are hollow inside with sloping spikes toward the distal end . There are no holes or pores on the hair and the irritating factor can only be obtained by crushing the hair . This suggests that when stinging occurs the hair is broken in the skin and the substance released (DUCOMBS et al., 1979; LAMP et al., 1982x). This factor increases the histamine content of various tissues (VALETTE and HUIDOBRO, 1954, 1956) and causes a reaction in guinea-pig skin, probably produced by mediators released by mast cells . Degranulation of mast cells by this substance has been shown to be dose-dependent, while heating greatly reduced these effects, suggesting that it is of a protein nature (LAMP et al., 1983) . In this study proteins from the hairs of Thaumetopoea pityocampa have been analysed by various electrophoretic and immunological techniques . These proteins were compared with cuticular and haemolymph proteins, and with those from the glands which produce 3" 17

348

MICHEL LAMY et al.

urticating hairs. Proteins were also analysed in urticating glands using immunocytological methods. The ability of each protein fraction to increase blood vessel permeability was assessed on guinea-pig skin . MATERIAL AND METHODS Extract preparation Thaumetopoea caterpillars at the last larval stage were used in this srudy and were collected on maritime pine trees near Bordeaux, France . They were maintained in the laboratory under the conditions described by L~tv et al. (1983) . Hairs were prepared from 77tatrmetopoea caterpillars at the last larval stage with an apparatus described elsewhere (DucoMas et al., 1979), which is similar to that used by Da Jotvc (1980) for collecting hairs of Euproctis chrysorrhoea . Hairs were broken in liquid nitrogen (PAPILLON and GASSIER, 1982) and crushed in

saline solution (0.9% NaCI). LAE~tMt.t (1970) buffer or Tris-glycine buffer (pH 8.6, 0.025 M) . They were mixed for 48 hr at 4°C and centrifuged for 30 min at 10,000 ~. The clear supernatant was employed fresh or frozen at -20°C. The protein content of the solution was measured according to the method of Lowav et al . (1931) . Lsemmli's solution was found to be the optimal extradant and when necessary the solution was concentrated by Lyophilisation . For dermatological tests the solution was initially dialysed against water for 24 hr . Thoracic cuticles of tergitea were carefully cleaned from adhering tissues, blood and alla with small pieces of wtton moistened with Ringer solution . Cuticitlar extracts were prepared as described for hairs. Larval haemolymph was collected into an ice-cooled test tube containing a few crystals of phenylthiourea to atop the melaniradon reaction . The haemolymph was centrifuged and the supernatant employed fresh or stored at - 20°C. Haemolymph was yellow at thebeginning of the larval stage and green at the end, owing to thesynthesis of bilatriene IXa pigment (CHOUSSY et al., 1973) associated with a moulting protein (LAtitv, 1971). Urticating glands were prepared after initial total depilation . They were washed and homogenized at 0°C with Laemmli buffer. Proteins were extracted with Millipore filters (Millex 0.22 pm) then dialysed against water, centrifuged and lyophilized. Antibody preparation

Hair extract was used as an antigen for the induction of antibodies by male rabbits. Hair extract (0 .5 ml) was supplemented with 0.3 ml of sterile Freund's complete adjuvant . The rabbit received four i.m . injections of 1 ml of each mixture for four weeks. After one week the injection procedure was repeated with 1 ml of hair extract solution, supplemented with 1 ml of Freund's adjuvant . The rabbit wss protected from anaphylactic shock by i.m . injection of promethazine hydrochloride (25 mg). live days after the leaf antigen igjection antibodies were revealed by OuctrreRwrrv'a (1%7) immunodiffusion test. Blood was removed from an earvein, allowedto clot, centrifuged and the serum recovered and stored in 500 ~1 aliquots at -20°C . This antiserum was thm adsorbed against wticular extract for 3 lu (3/4 anti-lotir serum + 1/4 cuticular extract) . After centrifugation (30 min at 10,000 t) the atpernatant was recovered and the procedure repeated at S°C overnight (1 volume anti-hair serum + 1 volume wticular extract) . The resulting mixture was aatrifuged again (30 min at 10,000 g) and the supernatant stored at 20°C until ttse . Immuno-hlstologkal methods

Immunofluoreaant staining was conducted by the indirect fluorescent antibody technique (Cooty dal., 1933) using an anti-hair serum adsorbed against cuticular extracu. Antiserum suitably diluted in veronal buffer (O .l M, pH 7.2) was incubated with tissue samples for periods varying as a function of the dilution . After washing, fluorraain isothiocyanate~ogjugated sheep anti-rabbit-Ig immunoglobulln was used to reveal the antiserum bound to sites containing the specific antigea. Evens blue (0 .019s) wee used as a counterstain to eliminate background staining and to obtain better contrast . Slides were scanned using a fluoreaoena microscope (Leitz Ortholux, lamp HHO 200, primary filters BG 12, saondary filter K 530) and the appropriate fields photographed on Ektachrome film . Immuno-djjJusion~t~sts (OucNrtaet .orrY, 1%7)

These were conducted on microscope slides coated with 1.554 agaroae. The center well contained antiaaum and the peripheral wells either extracts of hairs, cuticle or haemolymph, which were prepared as previously described. The immunodiffusion teat was conducted for 24 hr at 20°C . The gels were washed and stained with Coomaaaie Brillant Blue .

Urticating Protein of Thaumetopoea

349

Immuno-etertrophorrsis This was carried out on microscope slides coated with veronal-buffered agarose (pH 8.2, 0.03 M) as described by Gxweawx and WILLIAMS (1933) at 25 V/slide for 2 .3 hr at 20°C . The antibody is placed in long troughs cut out of the gel following electrophoresis . As a control, both long and short troughs were used in order to prove the identity of the observed precipitation arcs . Precipitation arcs obtained after 24 hr of diffusion were stained with 0.1 % acetate-buffered AmidoSchwarz IOB (0.1 M acetate buffer, pH 6) . Etectrophorcsis Electrophoresis in a polyacrylamide concentration gradient was conducted according to the method described by MARGOLIS and WRISLEY (1973), with a linear gradient of 8-30010 acrylsmide and Tris-glycine buffer (pH 8.6). Following pre-electrophoresis (1 hr, 100 V), electrophoresis was run at 4°C at a constant current of 150 V for 24 hr . Gels were stained for 1 hr in a solution of Coomassie Brillant Blue R 250 in SOOk methanol and 1001 acetic acid, and destained in a methanol plus acetic acid solution . Gels were stored in 7010 acetic acid . Gels were stained for carbohydrates and lipids, respectively, with Schiff periodic acid and Sudan B Black. Molecular weights of proteins were determined with the two following protein standard kits (Pharmacia) : a kit of low molecular weight proteins (20,100, 30,000, 43,000, 67,000, 94,000) and a kit of high molecular weight proteins (67,000, 140,000, 232,000, 440,000, 670,000) . Following electrophoresis on a 13010 acrylamide gel, the gel was cut and each fragment was applied under denaturing condition for a sxond migration (3 hr, 200 V) . For partial denaturation Tris-glycine buffer (pH 8.6, 0.023 M) contained 1 % SDS, and for total denaturation Tris-glycine buffer (pH 8.6, 0.023 M) contained 1 010 SDS and 2.5010 ß-mercaptoethanol . All samples in denaturing solution were heated at 100°C in a boiling water both for 2 min before the second electrophoresis . Dermatological tests Detmatological testing was conducted in vivo according to the method of OVARY (1%3). Control (physiological saline, Laemmli buffer, spinach extract) and test substances (total hair extract and isolated cuticular and hair protein fractions) were injected intradetmally into male albino guinea-pigs, as described previously (LAMP et aJ., 1983) RESULTS

(Fig. 1) A total of 16 proteins were detected in the hair extract by electrophoresis in the polyacrylamide concentration gradient . Seven fractions with molecular weights higher than 140,000 were at low concentrations and only a few stained with Coomassie Brillant Blue. The first three fractions, near the origin, are glycolipoproteins, since they reacted with Schiff periodic acid and Sudan Black B. Nine fractions with molecular weights lower than 140,000 stained more intensely with Coomassie Brillant Blue than those of the first group and are not associated with carbohydrates or lipids . More than 25 proteins were detected in cuticular extract . Those with mol . wt > 140,000 stained intensely only when cuticles were harvested from young moulting caterpillars . Those with mol. wt < 140,000 included the major proteins of cuticle. About 20 fractions could be observed . The seven fractions with the lowest molecular weights were absent in hairs. Fifteen protein fractions of high molecular weight (first group) (52,000-700,000) were observed in caterpillar haemolymph (last larval stage : LS). They were present in high amounts and dilution was necessary to obtain good separation. Comparative study of hair, cuticle and haemolymph proteins indicated that the high mol. wt hair proteins present some similarity with haemolymph proteins, whereas the low mol. wt proteins show similarity to the cuticular proteins . However, one protein (mol . wt 28,000) was observed to be present only in hairs. This protein was stained even when hair extract was diluted. This major protein, which is not associated with carbohydrates or lipids, was called thaumetopoein. Double electrophoresis of thaumetopoein treated with SDS showed two subunits of 13,000 and 15,000 mol. wt (Fig. 2). Analysis of the urticating Electrophoresis

350

MICHEL LAMY !r al.

gland showed that this structure has the same protein composition as cuticle, but also contains the additional thaumetopoein fraction . Immunologica! reactions Ouchterny's double diffusion tests with anti-hair serum produced continuous bands from the wells containing hair extracts, cuticular extracts and haemolymph (yellow or green) . One supplementary precipitin arc was formed with hair extract and was identified as thaumetopoein (Fig. 3). Immunoelectrophoresis confirmed these results. Four precipitin arcs were observed in hair extracts, three in cuticular extracts, but only one in haemolymph . Short trough techniques proved that one band of the hair extract was common to cuticular extract, another common to haemolymph . With an anti-hair serum adsorbed against cuticular extracts only a single precipitin arc appeared with hair extract in immuno-diffusion or immuno-electrophoresis and none was observed with cuticular extracts or haemolymph . The same precipitin arc is found' with the thaumetopoein fraction extracted after electrophoresis in polyacrylamide concentration gradient (Fig. 4). Using anti-hair immune-serum, with or without adsorption against cuticular extracts, we were able to demonstrate the presence of immunoreactive cells in the urticating gland . I

Ki ~

UH,

, H

iKZ

67 000 43 000 30 000

20 100

FIG. l . POLYACRYLAMIDE GEL ELECTROPHORESIS PATTERNS OF HAEMOLYMPH (H), URTICATING HAIR EXTRACT (UH) AND CUTICULAA EXTRACT (C) OF ThauMltopola pllyocampa CATERPILLARS .

Linear gradient of 8-309o acrylamide gel and Tris-glycine buffer (pH 8 .6) was used . Preelectrophoresis was carried out for 1 hr at 100 V and electrophoresis at 4°C at a constant current of 130 V for 24 hr . Proteins was stained with Cootnassie blue R 250 . KI and K2 are standard kits (Pharmacia) applied in the extreme left and right lanes of each gel . The arrow marks the position of the unique protein, thaumetopoein, in the urticating hair extract.

331

Urticating Protein of 77~arrmetopoea

1

-- 94 .000 -- 67 .000

,rrrr s~

25 .700 -

-- 43 .000 -- 30 .000

18 .400 14 .400 -12 .300 --

s

P T

FIO . 2 . DOUBLE SLflGTAOPHOAESIS OF THAIJMHTOPOBIN FRACTION OF TIIQIfInltOpOlIO pityOCOIIIpQ CAl'HRPII.LAIIS .

Following a fast decprophoreaia on a 139s acrylamide gd, the gd fractioaa were treated undo denaturing conditions and samples were submitted to a second migration. 1Proteln standard bu (BRL and Pharmacia) were applied in the extreme left and right lanes of the gd . The figure shows only the second decuophoresis . (A) Entire gd ; (B) detail of gd showing the dissoâation of thaumetopodn i~o the two subunits of mol. wt 13,000 and 13,000 . T, thaumetopoeia; P, another protein from hair extract of lower molecular weight than thaumetopeia, which has no irritating effect .

332

MICHEL LAMY d a1.

UH FIO. 3.

OUCFrrERLONY DOUBLE DIFFUSION PATCERNS OF HAEMOLYMPH (H!, URTICAI7NG HAIR Exrlucr (LJH) AND clmcvua ExTaACr (C) of 77+memetopoea pityocampa cwrEaPILLAAS .

Central well contains polyvalent antiserum against hair proteins (AH) Plates were allowed to develop at 20°C for 24 hr before being stained with Coomassie blue R 250. T indicates urticating protein, thaumetopoein .

Fia. S .

IMMUNOFLUORPSCENf SECTION OF A

Thaunutopoea pityocampa cwrsaPILLAn uancArINo aLAND.

Section through the abdominal urticating gland, treated with anti-hair immune serum adsorbed against cuticular extract, shows fluorescence in cells which produce the urticating protein, thaumetopoein . Bar scale = 10 lun .

Urtiating Protein of 77wumetopoec

353

A .h .c-

FIa .

4 . IMMUNO-ELECTROPHORBTIC PATTERN OF HAEMOLYMPH (H), URTICATn~O HAIRS EXTRACT (UH) Arm cvrICULAR EXTRACT (C) of Thaumdopoeo pltyocampa aTERPILhARS. Electrophoresis in veronal-buffered agarose (pH 8 .2, 0 .03 I~ with 2S V/slide for 2 .S hr at 20°C . The immunodiffuaion was conducted for 24 hr at 20°C with polyvalent antiserum against hair

proteins (AH) and with polyvalent antiserum adsorbed against cuticular extract (Ahc) . The precipiting ara obtained were Gained with aoaate-buffered AmidoSchwarz 10 B . Arrow indicates thaumetopoein . The bottom two immuno-electrophoresis runs used short troughs to show proteins common to urtiating hair extracts, haemolymph and cuticular extracts.

Immtmofluorescena disappeared when immune serum was pre-treated with hair extract. This negative control proves the presence of thaumetopoein in cells of the urticating gland (Fig. S). Dprmatological reaction to thaumetopoein Skin tests were observed 20 min after injection of test substances. A positive reaction was revealed by a blue spot following i.v. injection of Evans blue. A blue spot revealed an abnormal permeability of surface blood vessels following injection of a test substance. Total hair extract, thaumetopoein fraction and spinach extract, a positive control, gave positive reactions. Spinach extracts, as other plant extracts, gave a non-specific irritant reaction following intradermal injection (DE WECK, 1984) . Negative reactions wen observed with a total cuticular extract, protein fraction of hair extract other than thaumetopoein, acrylamide gel and physiological saline (negative control) . DISCUSSION

In as extract of the hairs of the Thaumetopoea caterpillar numerous soluble proteins were revealed using various electrophoretic and immuno-electrophoretic techniques . Some of these proteins are present also in cuticle and haemolymph. One fraction was

354

MICi~L LAMY et aL

found to be hair specific and we have called it thaumetopoein. Since hairs are part of the cuticular production of caterpillars, cuticular proteins form the major constituents of urticating hairs. Composition of cuticular protein in hairs was found to be constant during intermoult, while cuticle proteins exhibited some variations in concentration : they increased just before the moult phase and decreased during intermoult (CASS>EIt et al., 1980). Differences exist between soluble larval and imaginal cuticle proteins in many species: Periplaneta americana (Fox and MILLS, 1969); Galleria rnellonella (SRrvASrwvA, 1970, 1971); Pachynoda epphipiata (ANDERSEN, 1975); Terrebrio molitor (ANDERSEN et al., 1978 ; RoBERTS and WILLIS, 1980; Lemoine, Doctoral Thesis, University of Dijon, 1983); Locusts migratoria (PHILLIPS and LOUt:iH'PON, 1976). In this last species, LENStnt and CASSIER (1978) reported the existence of sex-linked proteins in the cuticle of adults by means of eloctrophoretic, immunodiffusion and immuno-electrophoretic analysis . One major antigen present in female cuticle, female blood and eggs was revealed by the antiserum against vitellin or vitellogenin (PAPILLON and CASSIER, 1982 ; CASSIER and PAPILLON, 1983). Cuticular differences between all larval stages were observed to be hormone dependent in Manduca sans (RIDDIFORT, 1981). Immunological techniques revealed the presence of haemolymph proteins in both Thaumetopoea cuticle and urticating hairs. This fact confirmed earlier observations of the existence of haemolymphatic pmtein in the cuticle of Periplaneta amerirnna (Fox and MILLS, 1 %9) and Manduca sans (KOEPPE and GILBERT, 1973). Radiotracer studies have shown that some blood proteins can be transported into the cuticle of Locusts migratoria (TOBE and LONt3HTON, 1%9), Manduca sexta (KOEPPE and GILBERT, 1973), Periplaneta amerltana (GEIC3ER et al., 1977) and Calliphora erythroaephala (ScI~rnLr~?R et al., 1980). In the last species, calliphorin, a fat body protein, was incorporated into the integument and was an essential protein of the tanned cuticle. In Locusts migratoria, the presence of vitellogenin-like protein in the cuticle depends on the presence of vitellogenin in the haemolymph (PAPILLON and CASSIER, 1982). However, luciline of Lucilia (THOMSON et al ., 1976), drosophiline of Drosophila (ROHERTS et al., 1977) and manducine of Mandata (Riddifort and Scheller, unpublished observations) are able to be used for cuticle formation. The various techniques employed here demonstrate the existence of a spa:ific protein fraction in caterpillar hairs which has urticating properties and which we have called thaumetopoein . The molecular weight was approximately 28,000, formed by two subunits : one of 13,000 and the other of 15,000. This protein appears to be very different from the Thaumetopoea wilkinsoni toxic proteins described elsewhere by Z>pROwSxx and ROLANT (19fî6) and identified as albumin and a~, ß and y globulins. The study of possible enzymatic activity of thaumetopoein in comparison with the venom of Euproctis caterpillar spicules (DE JoNO et al., 1982; BLEUMINK et al., 1982) is in progress . We have previously demonstrated a dermatological action of the hair extract of Thaumetopoea caterpillars in the guinea-pig (LAMP et al., 1983). In this study we confirm that thaumetopoein has an identical effect while other tested hair proteins were without effect. REFERENCES ArmP.ns~+, S. O. (1975) Compariwn betwan the sclaodmdon of aduh a~ larval cutick ln SchiAOCaro ~rr8arla. !. Insect P6yaioJ. 1!, 1603 . Armî , 3. O. (1978) Cuticular ~claodution in the balla Phrhynode epphiplata and Ter~rlo moJltor. J. Insert Plryalol. 21, 1225 .

Urticating Protein of T7zaumetopocx

355

Awame.~z, S. O., Czt~ae, A. M. and Wtt.us, J. H. (1973) The amino add composition of cuticles from Terrrbrio moUtor with ePec+al reference to the anion of juvenile barman . Insect Blocherm . 3, 171 . BIEUMINC, E., De Jowa, M. C. J. M., KAw~Matn, F., Mt~e, G. T., Kt.oosrmttrtns, A. and St .nJrmt-P~t., I. J. (1982) Protease activities is the epide venom of Euproctis caterpillars . Taxman ~, 607. Cesstme, P. and PnP1t.LOw, M. (1983) Le dimorphisme sexuel des protéines cuticulaires du criquet migrateur, Lotrrsta migrxtoria mlgrxtorlo~er (R d F) . Can. 1. Zool. 61, 2976 . C~ssmze, P., POac~aow, P., Pertu.ow, M. and Lews=r, Y. (1980) Contribution ït l'étude den protéines cutictrlarres du aiquet migrateur. bercera mtgmtoria migr~atorioldes (R. eL F) données quantitatives . Arms Scr. nest., Zool . 14° aErk 2, 51 . Cxoussv, M., LeaaouQuEae, J., BASama, M. and LMtx, M. (1973) La protéines de l'hémolymphe de la procesion_m"`n du pin (7lzaumetopora pitJwcampa Sdtiff). Etudes des pigments associés aux dzromoprotänes. C. r. hebd. Séant. Aced. ScL, Ports 277. 365. COONS, A. H., Lenuc, E. H. and CONNOLLY, J. M. (1953) Studies on antibody producxioa. I. Method for the hietochemical demonstration of specific antibody and its application to the study of hyperimmume rabbit . J. earp. Med. 102, 49. D~toz.na, ß. (1%3) Les miroir urticants de la prooessionnaim du pin. Revue Zoor. agrlc. appl. 10-12, 107. Ducoz~s, G., LMtx, M., Beaa~w, J. J., Tw~a"s®t, J. M., Geav~ts, C. and Textes, L. (1979) La chenille processionnaire du pin d l'homme. Btude morphologique de l'appareil urticant . Engines Épidémiologique. Arms Drrm . VLnH. 1"6, 769. Duooz~, ß., LAMY, M., Mot.t.~au, S., GUILLAAD, J. M. and Mwt,evtt.t,e, J. (1981) Contact dermatitis from Processioael Pie caterpillar (T7~rmetoPoea pltyoaampx Schiff. Lepidoptera) . Contact dermatitis 7, 287. Fox, F. R. and Mtt.ts, R. R. (1969) Changes in haemolymph and cuticle proteins during the moulting processin the American oockrwch . Comp. Blochern. Physrol. 29, 1187 . Gesses, S. ß., KROU, S. H. aztd Mtt.ts, R. R. (1977) Possible involvement of cockroach haemocytes in the storage and eyntheaie of cuticle proteins. 1. Insect Physrol. X30, 227. Gawa~a, P. and W~ , ~"uc , C. A. (1933) MEthode permettant l'Étudeconjuguée des protéitée ElectrophorEtiquee d immunodzimiques d'un mElange de protéines. Application au sérum sanguin. Biochrm. brophys. Actes 1", 193. Dz JoNa, M. C. J. M. (1980) A safe and rapid method of wllectirrg the urticating hairs from Euproctis caterpillars (Lepidoptera, Lymantrüdae). Crin. exp. Drap . 9, 357. De Iowa, M. C. J . M., KwwMto~ro, F., Bt.suMn~rc, E., Kt.oosrsaxu>s, A. J. and Meurt, G. T. (1982) A comparative study of the apicale venom of Euproctis caterpillars . Toxicon 20, 477. Koro'rs, J. K. and G1LaFJtT, L. I. (1973) Immunochemical evidence for the transport of haemolymph protein into the cuticle of Mandata sana . 1. Insert Physrol. 19, 613. t- "~~, U. K. (1970) Clevage of structural protdns during the assembly of the head of baaeriophage T4 . Nature 227, 680. Lwntv, M. (1971) Variation cyclique en rapport avec la mue larvaire d'une fraction protéique de l'hémolymphe de la prooesaionnaire du pin. C.r. hebd. Stanc. Acad. Sci., Ports 273D 2298 . Lev, M. (1977) La chenille processionnaire du pin et l'homme. Revue bibliographique. Bull. Soc. lion . Bordeaux VIIII, 25 . LMnr, M. and t-aa."rna, CI. (1983) Contribution il la systématique den ffylesia: Etude su microscope élatmnique à balayage den "flMhette" urticantes . Bull. Soc. ent. Fr. 88, 176. LMnr, M., Dueot~s, G., Pesrttne~w, M. H. and VtNCewnz=AU, P. (1982x) Productions tEgumentaires de la proc~onnaire du pin (77Yaunretopoea pltyocxmpa Schiff.) (LEpidoptères): apppareil urticant et appareil de ponta Burl soc. zoor. Fr. 107. SIS. Door, M., Mtt~t., M., Pze~ntNwun, R., DucoMes, G. and Mwt.evtt.t .e, J. (1982b). L'appareil urticant des papillons hlyltsta urtkoru Ft.octr a AHONNENC et H. umbrvta Schaue, rraponeables de la papillonne en Guyane française. Int. !. Inmt Morph. Embryol. 11, 129. LMnr, M., Vnvcetvne~u, P., DueoMZZS, G. and P~sruae~un, M. H. (1983) Irritating substance extracted from the 77~aumetoporx pltyocampa caterpillar: mMz, ..~zx . of action. Exprrirntra 39, 299. LMnr, M., Pesntae~uu, M. H., Novnrt, F. and DucoMns, G. (1984) Papillons urticants d'Afrique et d'Amtrique du Sud (g .Anaphar es g.Xylesla). Contribution il l'étude de leur appareil urticant er à leur mode d'action . BuU. Sot. soot F}. 109, 163. Lt+rrotY, Y. and C~sstzae, P. (1978) Contribution il l'Étude physicochimique et immunologique den protéines cuticulaires du criquet migrateur l.octrsta migrxtorla mlgratorloides (R . et F.) . C. r. lrrbd. alone. Aead. Sci., Aoris 287, SIS. Lowav, O. H., Rostaaaouatt, N. J., Fear, A. L. and Rwrm~t.t ., R. J. (1931) Protein measurement with the Folio pheaal reagent. J. blot. Clam . 193, 265. Meaaot.ts, J. and Wntst.ev, C. W. (1975) Improvement of pore gradient dectrophoresis by inaeaaingthe degree of cross-linking at high acrylamide concentration gradient . Brocheur . Brophys. Res. Commun. 27 . 68. Oercxrear.oNY, O. (1%7) Immunoàiffusion and immuno-elaxrophoresis. ln : Handbook of ExpMmentol Immunology, p.65S (Wets, D. M., Ed .l . Oxford : Blackwdl Sdentiflc. OVARY, Z., BaNecBtxeF, B. and Biocrt, K. J. (1%3) Properties of guinea pig 7S antibodies . Il . Identiircation of antibodies involved in passive cutaneous and ayatematic anaphylaxi:. J. exp. Mad. 117, 931 .

336

MICHEL LAMY et al.

PAP~LLOPt, M. and Cwss~e, P. (1982) is vitellogenin a cuticular component of the female locust . F.rrperienrle 38, 1476 . Ptnu .n~as, D. R. and LouoaroN, B. G. (1976) Cuticle protein in Locusts migretorle. Comp . Biochem. Physiol. 55B, 129. RIDDIFORT, L. M. (1981) Hormonal control of epidermal all development . Am. Zool. 21, 731 . RoaeaTS, P. E. 8IId WILLIS, S. H. (1980e) The cuticular proteins of Tenebrio molitor. I. Electrophoretic banding patterns during postembryonic development . Drvl Btol. 73, 59. RoeeaTS, P. E. and Waus, S. H. (19806) The cuticular proteins of Tenebrio molitor, II . patterns of synthesis during poatembryonic development Drvl Bfol. 73, 70 . Roasters, D. B., WotFe, I. and AewAr, M. E. (1977) The developmental profiles of two ms~jor haemolymph proteins from Dr»snphthr melenogaster. J. Insert PltysioL 23, 871. Scttet.teR, K., ZtsrntF.tu+wtv, H. P, and Stoats, C. E. (1980) Calliphorin, a protein involved in the cuticle formation of the blowfly, Celllphoro vicina. Z. NeturJ. Ser. C 35, 387. Swvwsrwvw, R. P. (1970) Electrophoretic behavior of cuticular proteins of different developmental wtges of Gallerie mdlonella 1. Insert Physiol. 16, 2343 . Swvwsrwvw, R. P. (1971) The amino acid composition of cuticular proteins of different developmental stages of Gelkrla mdlondle. 1. Insert Physiol. 17, 189. THOMSOPI, J. A., Rwnott, K. R., Sttww, D. C., WHrrrav, M. J., Fosrsa, G. G. and Btnr, L. M. (1976) Genetic of Lucilin, a storage protein from the sheep blowfly, Ltecilia cuprina (Calliphoridae) . Blochem. Genet. 14, 143. Toes, S. S. and Louottrotv, B. G. (1969) An autots+diographic study of haemolymph protein uptake by the tissues of the fifth mater locust . J. Itrsroct Physiol. 15, 1331 . Vwt.~rrs, G. and Hutt~oeao, H. (1954) Pouvoir histamino-llbErateur du venire de la chenille proaasionnaire du pin (77taumetopooe pltyorempe Schiff) C.r. S6a~. Soc. Biol. 148, 1605 . Vwt.erre, G. and Hutnoateo, H. (1956) Pouvoir histamino-Gbératew du venire de la chenille processionnaire du pin (77teumetopoee pityocampe Schiff) Cr. Sefonc. Soc. Blol. 150, 658. os Wt=.cx, A. L. (1984) Pathophysiologic mec nt "~+ " of allergic and pseudo-allergic reactions to foods, food additives and drugs. Ann. Allergy 53, 583. Zttmtcowsn, L. and Rot.wrrr, F. (1966) Study of the totdn from the poison hair: of T7tatrmetopoea wl/kinsoni caterpillars . 1. invest . Dorm . 46, 439. Ztrartowstt~r, L. and Rouir, F. (1972) Study of the LOJdn from the poison hairs of T7tatrmetopoen wilklnsvni. caterpillars . 1. invest. Dean . 58, 274.