TThhee LLeepipdiopdteorpoltoegircaollSoocgieitycal Society ooff JJaapapnan ueLv aTrans. tepid S.oc. Jitpa n58 (2) :172-176, March 2007 Scanning electron microscopic observations of the peritrophic membrane in silkworm (Bombyx mori) Iarvae Wataru MiTsuHAsHi* RitsukoMuRAKAMI and Nationa lInstit uotfe Agrobtologica lScicnces, Tsukuba, ibaraki ,305-8634 japan Abstract The peTitrephi cmembrane of the third insta lrarva cof a hybrid strain of thc silkworm Bomb.v. xmori, C146 ×Nl37, was observed by scanning electron microscopy. Thc cctoperitrophic network laye rwas well embedded in a protein matrix, suggesting tha tthe peritrophi cmembrane plays an important role jn protecti ntghe midgut cells from infectio nwith Bomb.vx mori nucleopoly- hedrovirus, Key words Entomopoxvinis, spind]e, nucleopolyhedrovirus, midgut epithelium, Anomata euprea, infectivity. enhancement of Introduction The peritroph imecmbrane (PM) is a non-cellular membrane that lines the midgut lumen of insect sin the fbrrn of a tube, extending from the anterior midgut to the hindgut (Derksen and 6ranados, 1988), The PM is composed primaril oyf chitin and proteins ,which include glycoprotei nansd proteoglycans (Wang and Granados ,2001), The PM protect sthe midgut epithelium from mechanical damage caused by ingested materials and exposure to toxins (Sarau eet rat., 2003) .It may also regulate the passage of materials int oand out of the ec- toperitrophic space and may compartmentalize the digesti vperocesses ,thus aiding the ethcient sequential breakdown of ingeste dmaterials (Sarau eet ral,, 2003) ,Moreover, thc PM plays a role in the protecti onof the midgut epithelium against attack by some pathogens ,including some insect viruses (Wang and Granados ,1997, 2000, 2001; Peng et at,, 1999; Sarauer et at., 2003) .For example, the PM is a barrie ragainst the approach of nucleopolyhedroviruses (NPVs )of the famil yBaculoviridae ,towards the microvilli of the midgut cells, which are the initi asi]te of infecti obny the virus in some lepidopter ainnsects (i e., some species of the famil yNoctuidae and a species of the family Arctiida e()Wang and Granados,1997,2000,2001;Peng 1999), et al., There have been few electron microscopic observations of the morphology of the PM in the silkworm Bomb)tt mori (fami lByombycidae) ,despite the estimation tha tthe PM in the gpecies could have the importan troles mentioned above (Rao et at., 2004), Our special concern is the degree to which the PM in B. }nori larvae is protecti vaegainst Bombyx mori NPV (BmNPV), B, mori is susceptible to BmNPV, and the infectiv iotfy the virus is markedly enhanced in B. mori Iarva e(hybr istdrains Nl37 × C146 and C146 ×Nl37; race Habataki )by proteinaceo upsaracrystall istnreuctures, called spindles, that are produced by an insec tvirus, the Anomala cuprea entomopoxvirus (AcEPV) w,hen BmNPV and the spin- dle swere co-administered orally (Mitsuha seth ial., 1998; Mitsuhashi and Miyamoto, 2003). Thus the spindles are potentia lsynergists of NPV insecticid esM.itsuhash iand Miyamoto (2003 )showed tha tthe PM of B. mori (hybri sdtrain C]46 xN137) larva ewas dramatically disintegrate dby perora] administration of AcEPV spindles. This strongly sug- gested that the disintegrat iofon the PM induced by spindles is the main cause of the spin- "Corresponding author, E-mai1: [email protected]; Fax: +8]-29-838-6028 NII-Electronic Library Service TThhee LLeepipdiopdteorpoltoegircaollSoocgieitycal Society ooff JJaapapnan Peritroph iMccmbrane of Bomb.)x mori 173 dles' enhancement of BmNPV infecti oinn B. mori. The enhancement should occur because the disintegrat iofon the PM and thus the reduction of its elfectiveness as a barrie ragainst NPV virions enable more BmNPV virions to reach the microvillus membrane of the midgut cells than when the PM is normal. However, the normal PM in B. mDri larvae was not ob- served by electron microscopy in the study by our group (Mitsuha asnhdi Miyamoto, 2003). Electro nmicroscopic examination would enable us to elucidate the pore sizes and their fre- quency of occurrence on the PM and thus to estimate to what degree the normal PM in B. mori larva eprotect sagainst pathogeni cmicrobes, includin gBmNPVI A PM that harbors larger pores tends to allow insec tviruses to pass easi]y to the midgut epithelium. Because some species have large-pored PMs, the PM is not considered to play an important role in protectio onf the midgut epithelium from infecti obny some insect viruses, includin gsome NPVs ([fana adnad Kaya, 1993; Mitsuhashi et al., 2006) .Rao et al. (2004 )showed by scan- ning electron microscopy (SEM) tha tthe pores in the PM ofB, mori larvae are smaller than those of insect species whose PMs are not considered to act as barrie rasgainst insec tvirus- es, at leas tat a certain ]eve l(rlana danad Kaya, l993; Boucias and Pendland ,1998; Mitsuhash iet al., 2006) ,suggesting that the PM at leas tconstitutes a certain leve lof barrier against insec tviruses, Nevertheles sRa,o et al. (2004 )did not disclos tehe name of the race that they used, and their observations were not made at high magnifications. No reports have demonstrat tehde pore size or distribut ioofn pores on the PM in B, ntori ([[br r2a00, 1 ). Thus, a detaile adssessment of the leve lof the protectio nprovide dby the PM in this species has not yet been made, Therefore i,t is significant to conduct electron microscopic observa- tions on the PM in the B. mori race used by Mitsuhashi and Miyamoto (2003 a)t various magnifications, includin gones higher than those used by Rao et ai, (2004 t)o, estimate the leve lof protecti opnrovide dby the normal PM in B, mori larva eagainst BmNPVI Here, we presen tthe results of our SEM study on the structure of the PM in a hybrid strain of B. mori larvae and discus sthe protectiv erele of the PM against infectio nby BmNPV, Materials and methods Silkworms We used a hybrid strain ofB. neori.. C146 ×N137, which has been maintained at our insti- tute .The 1arva ewere reared on aii artificial die t(Silkma Ntieh;on-nosan Co. Ltd, [[bkyo, Japan) 25eC at until use. SEM of the PM of B. mori Four larva eat the firs dtay of the third insta wrere individutt 1tr1aynsferre dto wells of six- well plate s(MS-8006 RS,umitomo Bakelite Co, Ltd, [Ibkyo ,Japan) ,on which small pieces (eac phiece 3 ×5 ×2 mm) of an artificial die t(Silkma thaed) been placed (on epiecelwell). After 36 h (i e.. on the third day of the third instar) t,hree larva ethat had fed completely on the diet were placed for 20 min in cold water containing crushed ice to paralyz ethem by chilling. The same treatment was given after 5 h (i e., on the firs dtay of the third insta rt)o one larv tahat had fed on only a litt lofe the diet ,Using a pa{r of forceps t,he PM was care- fu11 ydissecte fdrom each 1arv ain PBS (phosphate-bu sfalfinee;r eOd.Ol M Na,HPOsNaH, PO,, O.15 M NaCl) or sterile distille wdater, rinsed in O.1 M sodium cacodylate buffe ra,nd then fixe din 1 % glutaraldehyd ein 0.1 M sodium cacodylate buffe r.The sample was dehy- drate dwith an ethanol series (50%, 70%, 90%, 100%), and the ethanol was then replaced with isoamyl acetate, The resulting samples were critical-point dried using a dryer (JCPD- 5, JEOL, rlbkyo, Japan) ,sputter-coated with osmium using an osmium plasma coater (NL- OPC80, JEOL DfYI-UM, Tbkyo, Japan) ,and then viewed by SEM (JSM-6301 JEEOL). NII-Electronic Library Service TThhee LLeepipdiopdteorpoltoegircaollSoocgieitycal Society oofJfap anJapan 174 Wataru Mn'suiiAs Haind Ritsuk oINIuRAKAts･i[ Fis. 1.Scanning electron micrographs of the peritrophi cmembrane (PM )efBomb)'x tnori thjrd in- star larva c,A-C: the PMs of 1arvae at the third day of the third instar ,D and E: those of a larv aat the firs tday of the third insta r.All are the ectoperitrophic surfaces, Most of the porcs were smaller than 30 nm. As shown in A, a striated structure was found when the PM was observed at relatively low magnifications. In the PM shown in E, part of the ectoper- itroph inectwork layer has been exposed by the loss of protein matrix in which i tis embed- dcd. This ]oss of protein may have occurred in the coursc of preparati oofn the PM samplc for SEM. The innc rnetwork inside the pore sis indicated by the an'ows. The pores of the exposed ectoperitrophic network yary in shape and size, Results discussion and All the PMs examined by SEM were very similar. Numerous pores were observed on the surface of the PM, most smaller than 30 nm (Fig s]B-D). Thus, the PM in this race harbors far smaller pores than those in larva eof some insect species describe dabove whose PM does not play an irnportan trole as a barrie ragai'nst NPVs. The pores were located in the protein matrix in which the ectoperitrophic network iaye rwas embedded (Fi g1.) ,BmNPV virions are 35-45 × 300-330 nm in size (Hukuha r1a9,79) ,The network insid ethe protein NII-Electronic Library Service TThhee LLeepipdiopdteorpoltoegircaollSoocgieitycal Society ooff JJaapapnan Peritroph iMcernbrane of Bomb))x mori 175 matrix was multilayercd, as also reported by Yamazaki (1955 ()Fi g.IE) . Therefore, BmNPV virions were not considered to pass readily throug thhe PM, These structural find- ings strongly suggest that the PM function sto a considerable degree as a bamier against NPV virions. However, increase damounts of some BmNPV-gene products (e .g. en- hancin) ,could disrup tthe structure of the PM and thus allow greater numbers of bac- ulovirus virions to pass through the PM; thus, the intak eof an increase dnumber of virions into the midgut would diminis hthe protecti vreole of the PM (Lepo rete al., 1996; Wang Granados,199Z 2001). and There was a ditferen cien the surface of the PM between the larvae at the third day ef the third insta arnd the larv aat the firs dtay; the PM in the latt earppeared thinne rand it ssurface appeared rougher. The network within the protein matrix was more clearly detectabl esi,nce there were large rbulges in the protein scovering the network (Fig ]sD and E). This differ- ence may have been due to the differen cien age of the larva eafter the second ecdysis. The pores of the network varied in shape and size, and a small proportio vnvas hexagona lor circular (Fi gI.E), while these shapes are commonly observed in those of the larvae of the coleopteran species, A, cLgprea (Mitsuha est hali., 2006). The species B. mori i' sdivide dint oa number of races, and it ssusceptibility to BmNPV varies among these races (Furu t1a9,94, 1995) .Therefore i,t is possibl ethat the ultrastruc- ture of the PM varies among these races with respect to pore size (an dhence susceptibility to BmNPV). It would be interestin tgo examine the PMs ofraces other than the one used in the presen tstudy. Our resu]ts strongly suggest that the PM in B. mori (C14 6× N137) can protect against BmNPV to a considerable degree ,By using another method such as flu xmeasurements (Pen get al., 1999) ,it will be possible to demonstrat ein detai o1ur conclusion that the PM in th eB. mori race plays an importan trole in protecti oangainst the passage of BmNPV through the ectoperitrophic space, Acknowledgement We thank Ttikas hHiattor iof JEOL DMUM, [Ibkyo ,Japan, for his technical support with the SEM. References Boucias, D. G. andJ. C. Pendland, 199g .Insect-patho greelnationship. bt Boucias, D. G. andJ. C. Pendland CEds )P,rincipte sofIns ePact'hotlogy 1-3e. Kluwer Academic Publishers ,Norwell, Massuchusetts. Derksen, A, C, G, and R. R. Granados, 1988. Alteratio nof a lepidopteran peritrophi cmembrane by bac- uloviruses and enhanccmcnt of viral infectivi tVyir.ology 167: 242-Z50. Furuta, Y,, 1994. Susceptibili toyf lndian races of the silkwonn, Bomb>'x mori, to the nuclear polyhedrosis virus and densonucleos viisruses. ilcta seric. Ent, 8: 1-10 (j nJapanese). , 1995. Susceptibi]i toyf the races of the silkworm, Bomhy･x mori, prescrve idn NISES to the nuclear polyhedrosi svirus and densonuc]eos viirsuses. BuU, natn, bist .seric. ent. Sci .15: 1]9-14 5(i nJapanese with English sumrnary). Hukuhara, T., 1979. Insec tPatholog.y 218 pp. Japan Scientifi Scocieti cPsress T,okyo. Cl nJapancse). Lepore, L. S., Roelvink, P, R. and R, R. Granados ,1996. Enhancin, the granulosi svirus protein that faeili- tates nucleopolyhcdrovirus (NPV) infection si,s ameta]loprotease. J. Inverteb rP.ath .68: 131-140. Mitsuhashi, W,, Fu}'uta Y,. and M. Sato ,1998. The spindles of ttn entomopoxvirus of Coleoptera (Anomala "rpreaJ strongly enhance the infectivi toyf a nueleopolyhedrovirus in Lepidoptera (Botnb ymoxri). J. bivertebr .Path ,71: 186-188. Mitsuhash iW,. and K. Miyarnoto, 2003. Disintegratio nof the peritrophi cmembrane of silkworm larvae due to spindles of an entomopoxvirus. J. Jnverteb rP.atl i8.2: 34-40. NII-Electronic Library Service TThhee　 LLeepipdiopdteorpoltoegiroaollogical　 SSoooiceityety 　ooff　 JJaapapnan 176 Wataru　MITsu／HAsm 　and 　Ritsuk　oMuRAKAM 【 Mitsuhashi，　W ，，　Murakami ，　R．，　Takemoto ，　Y．，　Miyalnoto，　K ，　Wada ，　S．　and 　H ．　Kawakita，2006．　LllecLro　nmi − 　　croscopic 　observations 　of　lhe　peritrophi　cmembranes 　in　the　cupreous 　chafer 、　An‘丿mala 暇ρ惚 α，乏uld　the 　　silkw ・・in，　B・mb ｝・川 ・ri・茄 ・t・・57th　Meet ，κ・nt・B・α励 ゆ ，　seric ．　S・1．5・・∵ 14（in亅apa ・c・e）． Peng，．L，　Zhong ，　J．　and 　R、　R．　Granados，1999．　 A　baculoviru　senhancin 　alters　the　permeabilit　yof　a　tnucosal 　　midgut 　peritrophi　cInatrix 　from　lepidopteran上arvae．ノ．∬nsect 　Ph｝，siol．45：159−166． 　Rao　，　PRe．n，　lFliaancdcrha，i，　2oL00り4G．　iAocrMdNaPnVa，　B　，C，　hEigAu　iprloeto，e　riMn　d．i，　sCrounpgtisut，h　Te　p，e，　rBiutrrlionpi，　h　mNiemc，b，　raAnercie　lanl，　doS　a．，l　tCerosr　mriadgduto，　G　p．　hayndsi　oFl−． 　　ogy 　of　Bo〃zわyκntori 　larva，cノ｝zsclct　biochem．　motec ．βごol，34：12〔〕5−1213， 　TTSeaarr　nraaaund，l　eaaWrt，，　　riYBx　．．R　　aLo．nfりd2　0t　GhH0ic1l　．　d．1K　iQa．T　t，Klht　aCne〔）yn　，　aodarbn，iadgc1ik　9nD9　　3．a　mnHo．　detIhg　nfe，sd　uePuncs1ct廨　1t，）ai2l〃0ohao0　nl　o3oSsf8．y　　』ttCh）oh，e，s　aitr6nae6scl6e．t　　lcpeIat　rpnpi．s　ezeAracoitt　iamtood　rloneeofclp　anh　n．　mii　B　eicPmc’bn1or’eat’sn，cse1s，　2tN：ie3　w　n3laan3nu　dlcY−io3　npr4ek　3fr．．rlotmr　othpeh　　pgieecrl．i　しroAprhiech． 　　ノnsect 　bioch〃e！．1）hvsiol．47：47−61． W　an　gP，　’・Poc，　．a　lnlatdn　R、．A　Rca．　dG．ra∫dna，dσos．　，S．1　A9．9974．　：A6n9　7i7n−t6e9s8t2i．n］．a　i皿ucin 　is　the　target　substrate 　for　a　baculovir　uesnhallcin． Wang ，　P，　and 　R．　R，　Granados，2000．　 CalcoHuo　rdisrupts重he　midgut 　defensc　system 　in　insecl，　sInsect 　　bioc／hem．　molec ，　Bioi．30：135−143． 　　　 ，2001．M 〔｝1ecu1肛 structure 　of　the　perltroph　miecmbrane （PM ）：identificatj　oefn　potcnti　aPl．V［　target 　　sites　for童nsect　coDtrQl ．　Arch．　Insect　bi‘）（theJn ，ノ）hvsiot，47：llO−118， Y　am　azSatkn　i1｛，　DH：2．6，91−935355，　（Sitn　uJdaipeas　onne　st　hewei　tphe　rEintgrloips　hh　smuimemcmabrryanc）．　of　lepidopter　oiunssects．　Bu〃、　Nとig ‘tno　s（tric ．　exp ， 摘 要 ・ 走査型電了顕 微鏡によるカイコ幼虫囲食膜の観察 （三橋　渡 村上理都子） カイコ幼虫囲食膜の竃于顕微鏡レベ ル での観察報告がほとん どない ため に，昆虫ウイルス等病原微 生物の同虫へ の 感染成立お よび同虫品種問での カイコ核多角体病ウイル ス （BmNPV ）へ の感受性の 差異における囲食膜の重要度は不明である，この ため，カイコ交雑品種 （C正46×N ！！37）3齢幼虫の 1井「食 ・ 膜を走査型電了顕微鏡で観察し，BmNPV 等の 囲食膜通過の 難易 を検討した．皮膜細胞層側表而はタ ンパ ク層がよ く発達してキチ ン編み目状構造の孔をほぼ埋めてい るために孔が小 さく，この発達が悪 い ため に孔が大 きくて ウイル．ス通過が容易とされる一部ヤ ガ科等昆虫の もの とは明確に異なっ てお り，BmNPV に対 してかなりの程度の防御能を有する もの と考えられた．脱皮後の 「ヨ齢が 0 日の幼虫の 鬪食膜で は3H の もの に比較して皮膜細胞層側の表面の タンパ ク層が薄い と思われたが，これが脱皮 後初期の カイコ幼虫の 一般的傾向で ある可能性がある．また，キチ ン編み 目状構造の 孔の 形，大 きさ はコ ガ ネム シ の 種 で ある ドウガ ネブイブイ と比較 して変異に富んで い た， 〔Accepted　October　30，2006） Publtshed　hy　the　Lepidopter 10gica　lSoじiety　of　Japan， 5−20，Motoyokoyama2 ，Hachioji，Tokyo ，192−006．3Japan 　 　 　 一 NNI工I工-EElleoetcrotniroonic　 LLiibrbarryary 　Service