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Evaluation of stra;ns of Bacil1us thuringiensis as biological control agents. of the adult stages of the carrot weevil, Listronotus oregonensis (Coleoptera: Curcul ionidae)
6Y
Fabi en ne Eugen; e Joseph Saadé
Department of Entomology • MrGlll University, Montreal
fi t he~, I~, ~Ubllll tted to the ,- acul ty of Gr Sc l ence. (March 1993) (ll Fablenne E. J. Saadé, 1993 • rllJrne l ,dill 11111 1 Il,'('n ~t' _ Jo,,! pli :'ddd(. ______D'II.·rt(J',on ,1l-drfJds IfI'f'rll(J'IfJflnl 's CJrflJng._·d bl h'Ofld g,~nerol slJl)l~t categorres PleoJe select the onoè sub1ect wh,ch most r,' orly (kIl "f,..,', tl.,· ((",knt rJf 1"'Jr rJ,~',( rtlJtlon Errter the rorrpspondlng four digit code ,n the spoces proYIJed ) ';IIJ:l(jll)l. [ liJir--r3--'1~5-'-1 ---'31 U·M·I SUBan lf~M SU8JECT CODE Subjcd (ûtegorlcs THE HUMANITIES AND SOCIAL SCIENCES fOMMUNlfATIONS AND THf ARTS f fi, 1 .. 11 ()tJ /5 PHILOSOPHY, RElIGION AND Anc,enl 0579 f. 1 1.,. Ifi /'1 0') 3 ~ THEOlOGY M.,d,evol 0581 " l '1 (j',/? I.,f H, t JI t j/' ri ,l, ""f'hy 0422 Modern 0582 (, Il ,t J (l!14 I,t'UI r«IISJ1ûn Black 0328 IJ r ~ l J'U .. (1 .1' '. j l' ~ r ~ ~'H f~ It>fnl 0318 AIr,ran 0331 (, j Sl .. ~ Il (, , 'jc) '4 f '1 If' tI.f1·, Ml' (fll ~llrJ P:, 0321 AI'O, AVltrol,o n'lcl vc()n,o 0112 1) ')/ / ~ .... Il J4() 1111 Il'' rl,I,! " "li Il (l, '-JI 0319 (or,od'an 0334 rd,;l 'J',Je.; 1".,,1\.1 l" fi' 1 II Il "r, toI ()J20 r "'''f'''On 0335 1) ~'*'<~ 1, l' ~ , r T • J" f ,f J 1)', )0 l(J'I'") I,LIIJ' t (... 'I f f'f ,l, ,,,~hy rA n3L2 Ar.lf:r con 033// Mfl ((H'I" , ,1 (,,'(JH f ç f'l 1('l)t (J! 1() M,rJdlc ",le,,, "'J' Tf '..-"'0'3Y n46Q r 0133 M'J,li 'lA 1 -l l" t, ','1 Il.;,, 1 III' 'f r t., (i/fl8 U",t.'<.l 'jlotel 0337 {,t1 (.,; (J/47 "/)4"'" Il ( l,trin ' " ~ 'l' 'If'nl SO THE SCIENCES AND ENGINEERING II00OGI(AL SmN Suggested short title= Bactf~rJal control of the carrot weevil in Quebec • • • ABSTRACT M.Sc. Fabienne E. J. Saadé ~nto:nnlllqy Evalua~ion of strains of Bacillus thuringiensis as blo1ogical control agents Gf th~ adult stages of the carrot weevil, li\tronotus oregoncn~;~ (Coleoptera: Curculionidae) Strains of Baeil/us thunngiensis active a~JiTlst ColPOptp)\1 WPlt' evaluated for tOX1City aqalnst the adlllt c;talJP of tlw ( Listronotus oregonensls. Mortallty -'nd fras:, blOé\<;,dY<, W,lllq d '.tll LIIl\ l' sem;-artificlal dlet c;howed stralW; 1\30, 1\429 ,lnd BTl 10 hl' hlqhly lOXll Mortal ity r-erslsted aftu Hllt lal exposurr to th(, bdC tpr Id Wllh t Ill> SllrVlvors not re::'llf111 TlCj norn,dl f(>('dlnq I\ttPllIpt<, to YI'I',ol.dl' n, thunnglensls from the Insee te, ,'eveal('d [J. thunnglcnsl'> llk(> (HCjdlll'.rIl', III the gut dnd ln/on other structures I\t thp ITIldgut pit of ttH> 11I"!>ll (pli • 8.0), the crystals of the toxle straln'- wen! c;i~lnlficdnl Iy rnOlP ',()Iuhlp III vlL"o than were crystals of thp lp~'J toxi( ~trdin 1\311. P)o!polyt Il actlvation of the crystdls wlth gut extracts jleldpd d protplIl bdlld (bb ()7 kDa) for stralns 1\30 and A429 WhlC:h wa', slmilar tJ th!' dppdt'l>ril !IIo]('(Il],H welght of the toxln proteln for BTT fVldencc ',IlCj'JPsL', !hdt !hl' I(JW tOX1Clty of straln A311 mlght be due, ln pélrt, tn Uw db~('rl((> of 1 hl' !O/,I( moiety of the n-endotoxln . • RESUME • MSc. FabIenne E. J. Saadé Fntomologle l'évaluation de l'effet toxIque de plusieurs souches de Baci71us thur;ng;ens;s contre les adultes du charançon de la carotte, Listronotus orcgoncnsis (Coleoptera: Curculionidae) Pluc.I(!lJr', ~olJch!':; r!p nacil/us thunnglcnsls connues d'êtr':) actives c (ontrp 11". Colf!optere ) ont été rvalué pour leur effet toxique contre les ,HJlJlte~. du charançon dl' là caroU!", Listronotus oregonensis. C'est par le bldl~ de blOe\'.dl:" de mortallt(! et de matlere fecale qu'on montre que les '.ou ( hdr(Hl,OIl dp 1i-l carotte s(' nOUrl ~ sant Sdr une nourrIture semi - " u r v 1 Vcl Il t l' e f f e t t 0 X 1 q LI e d e ~ sou che') n' 0 nt pas reg a 9 né 1e urs hil b it u j e s ,t1lmpntdirp::. nO!'''',llps FaIt 'iurprenant, l'isolatlon de B. thuring;'ens15 <11", ddultf". à yevplp Id pres~nce dp mIcroorganismes apparen~és à B. • Ihllr/flg/clls/s ddW" II' tuhl' dIgest If de::. lnsectes dlnSl que cldns d'autre \tructul'es. A un pH COITf.'\ponddlll cl (plu] du tube dlgestlf dc's ddultes (pH i3 . 0) lIa s [) 1u b l lIt (, d(! ~ cry s t dU x des sou che S t 0x i que s é tait c,lqrllflcdtIVf'ment plw, plevee ln vitro que les crystaux (le la souche peu toxlquP .1\311. ['actlviltlon proteolyLlque des crystaux pdr 1es extraits du t Il tH' d 1q p~) t lf d do nrH' li n p Il and r pro tel n l que (6 6 - 67 k 0 a ) peu r 1 e S sou che s 1\30 pt 1\419. le pOld', moll'culdlre de cette bande était sirlilaHe à celUI d(' Id tOXIIll' protplr1HjUl' dl' 8T1 L'analyse protelnlque des ,ouches suggere qul' Id fdlblp toxlclte de la souche A311 est dûe, en partIe, a l'absence de la portIon tOXlqUP de la h-endotoxlne . • iv • • Oct! i Cd t ed to ,lot', ,JI'Illly dlld 1 ddy • ACKNOWLEDGEMENTS • wOllld Il ke to thank my co-supervlsar, Dr. G~ry B. Dunphy, for hlS '.Ilpport and qUHlanre and for the stlmu1atlng dIscussions regard'ng rJurnerou', a',ppcts of thl~ study. I also thank my co-supervlsor, Or. vil 11 iam rI YIllp, feJY hl', Pflfouragf!lTIent throughout the project. 1 am Indrbtf:d to Ur Roger BernIer for maklng thlS proJect possIble tJy IH(Jvldlrlfj Ihf' tJd(tf~r'1.]l stralns and useful technlcal InformatlOn. 1 W()tJld Il kp 10 0xprrss fTly grat Itudp tn the Department of [Iltomolagy Mid Ulf: ~dculty of Graduate Studles and Resl::arch for flnanCla1 suppod ln thf' fmm of ',rholdrshlps , tearhing assistantshlps and ')ummer research dWù rd', fhdnk', cll',(J t f1CjUP'. t Many Ihank", a'. well, ta PIerre Langlois for his technical help, anù t () Il 1 MI fi KIn Ci cl n ri r~ ,1 fl (> Ku bec kIf 0 r the i r sec r e t aria 1 a s SIS tan ce. Ihdnk') ln t\rnaurl Jdr];ln and Andrea Schroeder for taklng rarp of the l!l'd'lI ( u11 urp,> 1 .ll~.o thùnk !tIf> mpl1Ihl'r'-. of the farulty and graduate students of the • dfljltltll1tflnt of fnton101oqy who have cantnbuted to thé reallzatlon of this vJIH~ ln Pdrtllllldt', 1 dcknowlpdqr the frlendshqJ and vd1uable diSCUSSIons (lf I\dl.l Hdlwrlnl. rhdnks 10 Tanya Searle, especially for her aSSI stance wlllt tltt' Impct'" and to Grdham fhurston for hl"; good humour. Thanks a150 IjO III [lI Ilany .jpwpll for thp valuablp technlcal tips. l,lm cl 1sn l ndrbt pd to my parent s and une 1es for thei r encouragement .litt! nUl!'.! 1 ~,upport 1 1 Il cl Il y , wlsh to acknowledge the invaluable feedback, 111l1!<>!",ldndlnq, (,Uppot't and love, of my husbdnd, Edward Awad, throughout t Il 1', plO J t' ct . • vi TABLE OF CONTENTS • ABSTRACT 111 RESUME 1 \ ACKNOWLEDGEMENTS VI LIST OF TABLES \ LIST OF FIGURES '<.1 GENERAL INTRODUCTION Chapter 1 L ITERATURE REVIEW •...... , THE CARROT WEEVIL ..... Taxonomy and dIs t r !lJut lOn ~) Pest s tatus ~) Descnpt10n of stages Adul t E99 . • Larva Prepupd dnd pupa Life cyclp Damage lU Adu lt s 10 L arvae Il Types of control II Chem1cal control Il Cul tu ra 1 cont ra l If B1oloqlCal control 1/ BACILLUS THURINGIFtlSIS H1StOrl cal haci<'f)round Nomenc l ature SporulatlOn and cry',tal produrtHJfI Types of tox 1 nr, uther than thf: l, -f~nd()t(J/l ri u-exotOYln • VII B-exotoxln 23 6 -endotoxln . 24 • Mode of act Ion 25 REFERENCES 30 Chapter 2 Respons(~ of the carrot weevi l, Li stronotus o regonen sis (c'oleoptera:Curculionidae), to strains of Baci77us thuringicnsls. 39 ABsrRACT 40 INTRODUCTION 41 MAlERIAlS AND MtTHODS 42 Cheml ca 1 s 42 Bdcterial cultures and c;pore-crystal su spons l one, . . . 43 Colony and reanng conditions. 43 SelectlOn of dlet rnaxlmizing insect feeding 44 Insectlcldal Bloasc;ays .... 44 Insect survlVal and frass production after • rcmoval fram the diet containing ICP 45 DetectlOn of B. thunnglensls-like bacteria 46 Data analysl~ . 46 RESUL T5 ...... 47 [llet selectlOn 47 Determlnatlon of crystal protein content 47 lCP effects on 1 nsect morta 1 ity and frass product 1 on 47 Insect survlVal and frass product ion after removal From dlet contalning lep .. 55 Detection of B. thunnglensls-1ike bacteria 66 DISCUSSION 66 REFERENCES 71 LONNfCTING STATEMENT 75 • vi; i Chapter 3 Solublllzatlon and rroteolytlC proct?sslng of Bac7llus thu,·,n97t."\n~7\ • s-endotoxin ln the mldgllt fllllds of thr canot wl'l'vII. 11~t,\lnl'tu\ oregonen:515 (Coleoptera: CurcullOnldae). In Vltr'ù 16 ABSTRACT . . li 1NTRODUCTI ON !tl MATER l AL~ AND METHODS 19 lhemicals Il) Insects. . BD Bacterial str'alns BD Mldgut isolat Ion and dlgec;t IV\' flll Id pli Uo SolublllzatlOn of crystals at )electl'd pH v,lllll'\ BI Residence tlmp of food ln thp qut .... E1 e ct t· 0 p ho r P ') 1 S 0 f pro t Pd" (1 - d 1CI l") t (' ri (r y " LI Je, B(' Data analysis . il l RESUI TS . B3 pH measlirement of 1T1ldgiit dlljP',tlvp fl\ll COI~CLUSION AND FUTURE RESEARCH 100 APPENDIX: Source of Bacl llus thuringlC'n';l~ strdlrl', 101 • 1X LIST OF TABLES • Chapter 1 Literature reVlew Table 1. Dimensions of adult and egg stages of the carrot weevil, Listronotus oregoncnsis ...... 7 Table 2. Head capsule wldth of the larval instars of the carrot weevll, Llstronotus oregonensis ...... 9 lable 3. Insects evaluated for susceptibility ta Baci77us thurlnglensls ~ubspecles tenebrionis (= subsp. san diego) ...... 16 Table 4. Bac1l1us thurlnglensis subspecies tenebrionis-based commercial products .... 17 labll' 5. Host speclflclty of crystal proteins 19 Chapter 2 Tablp 1. The median lethal dose (LO so ~g protein/ml) for adu1ts of the carrot weevil, Llstronotus oregonensis, exposed to deslgnated Baci7lus thuringiensis strains for • 8 days at 25°( ...... 52 Table 2. DetectIon of Baci71us thuringiensis-like bacteria in/on dl fferent IrlSect structures and in the insect env 1 ronment ...... 67 Chapter 3 Tabl el. pH measurements of the midgut of the carrot weevil, Llstronotus oregonensis, fed selected diets • . . . .. 84 • x LIST OF FIGURES • Chapter 1 Literature reV1ew Figure 1. Diagrammatic scheme of sporulation ln 8. thuringiensis ...... Figure 2. Domains of activated Bacillus thuringiensis lUXln ?6 Chapter 2 Figure 1. The effect of food compositlon on aJult rdtp of defecatlon 48 Figure 2. Crystal-spore mixture protein content of different bacterial stralns 50 Flgure 3. (A) Dose-response curves of the> 1110st vlrulent strdlll'" A30, A429, and BH, compdreu to strdln 1\311 whicll Il,1', low vlrulence. (B) lorrespondlng prohlt rpqrp<,c,llHl 11 nes 53 Flgure 4. (A) Oose-response curves of Bac/llus thunng/ens/s stralns A30, A429, [3T1, anrl 1\311 Hl terme, of thl' • cumulatlve product lon of frass pellets over d fH'rlod of 6 days.(B) lorrespondlng regrrs~lun llnrs Sb Fl9lJre 5. The relatlonshlp behlern Hl., '1 vdllj(H) (do~f' IndlJc lnq SO'ic, de cre a sel n f ra:) <; pro cl LI C t 10 n) 0 f Ua c 11 1u ') t Il ur/ n 9 / r Wd " stralns 1\30, A4?9, 13TT, and 1\311 and thplr lorrp<,prHldlllCj LO'N values (medlan lethdl do,,!:) se Flgure 6. The effec.t of stralns A30, 1\429, 8TT, ùnd 1\31] of II. thunnglensls on the rate of df'fecatlon ln ri frd',', bioassay uSlng a representatlve dosalj(~ of nrJ/I(j proteln/ml 60 Flgure 7. Cumulatlve 7-day mortallty of thp adlJlt', of Listronotus oregonensls that had survived a rrlur 8 rjdy exposure to thE: L[j,,) do~age lCP of dl ffen:nt c, trd 1 rt', of B. thunnglensls 6? Figure 8. Rate of frass production of Listronotus oregonen,,/', previously exposed to LD~" va 1 les of Bac/llu', • xi thur/ng/enS1S straln A30, A429, 8TT and A311 64 • Chapter 3 f 1 CJ lJ rp 1. The solubillty of the crystals of different strains of Baci llus thunngiensis in universal buffer at se~ected pH values ...... 85 1 Iqurr! 7. R{jlatlonshlp betl-/een the percent solubililY of A429, BTT and A30 strdlns of Bacillus thuringiensis at pH 8.0 and thfllr LD~'I values agalnst the adult carrot w2evlls 87 f 1 çj II n' 3. fJroteHl content per cry:;tal of the dl fferent strains of Bac/l7us thunng/enS7S ln unlversal buffer (pH 8.0) .. 90 IlfjllYp4. Proteolytlc procr>sslng of the crystals of Bacillus thuringiensls stralns A429 (A), BTT (8), A30 (C), and fl311 (0) ln thl gut juice of the Listronotus oregonens/s. 92 • • xii -- ~------• GENERAL INTRODUCTION The carrot weevll, Llstronotus oregoncnsls (Le Conte). 1'- ,\ maJor pest nf several crop plants ln the northpdstern ~tdtL'~ of \tll' Ul1itl'd States of Amerlca (Chandler, 1926; Peppf'r. 1Q4? WI11ttOlllh. l'hl:') ,llId Canada (Perron, 1971; Martpl ct al., 1975, Stpvl'n~l)J1. \976) It ,lIt.lt~' Umbelliferou~ plants, but IL pleferred host I~. th\' ldlTot. [)llULLI~ ( In Quebec, the carrot WPE'vIl I~ lITllVOltilH' (Pl'lrol1. 1971) 1Ill' overwlntered adults lllvade calTot fIeld::, Irl the ~,prll1~! drill fl'l'd on thl' I1I'W foliage (Bolvln and BelaIr. 1989). Thr adult fl'IllJll'<' oVlpo~1t III tl1(· <,LilI< or crown of yOUflC) plants ,lnd thc' ldrvap, lIpOIl hdtdlllH!, pl'l\(\tldt!' th(' carrot roots. As ttlf' lan'i1e fl'cd thpy mdkl' tWlllI'I', III th!' lll'pl'I Ollt' tlllld section of the root ('ffplt lVE·ly Y'edUClnCj (rop VJluf> (Wh1t( o!1l11, I%J) Sin cel 967 1 edIT 0 t r 0 0 t l n J ur H' ~, ca lJ', (' cl II Y 0 r c <1 0 Il t' fi ., 1 \ Il tl V(' ranged from 2% to 22~; 1 n (llrebec (Pprron, 197]) Whl'l1 1fi ft ur\( Oll t 1 () 1 Il'd, th l s l n sec t pest ca n c d li Sl' d aIII d q (lof \l P t u 40 i, 0 f t tH' li II (' tJI' ( (cl 1 1 () t (1 () P ( BOl V ln, 1985 a) r e du Cl n 9 t Il e a c ( e p t il tl1 1 1t Y 0 f the l rOll t (J 1 h P Il d \ ~ 111 li cl Il d cannlng lndustrles whlch can tulf:rdte no Illon' thdll 5'X, root 111)111Y (l'PIlori, • 1971). The only recommended control nH'ihUI(', to (lctlf', l', Ill(' 11',(' ()f lnsecticldes dlrected agaHlst thp adult\. ltlf' prot>l('fI1', lnIlPI!'nt WIll! th!' use of chemical lnspctlCldes (I.e. enVlrOTln\('ntdl «(JllLlIllllldt 1011, 111'.('( t reslstance, effects on non-tarcjl'c orgd[II~,m',), ,Hld th!' h('!jITllllfllj of tHl lntegrated pest management (IPr~) proqrdll1 f(JI' (dtrot'. hdv(' !'tt(()Urdqf>r! research Into altf.?rnatlve lonlrol mPd',llr(l', f!f",Pdrch (OTI'.l',t(·rj of th!· potentlal use of an egg pard:.ltold, I1naphc', ,>ordldatu', dllrJ of thr(l(' speCles of entomogenous npmatode~, 5tclncrnrma carporap'>dc ~ f0/tldP (Polnar, 1989)], S fcltlao [- ) blblonl', (]J(JIlIdY, ICJW))], dftrl Heterorhabdlt15 bactenophora[- 1/ hcl/otll1dl', (fi01rldY, ](J90) 1 tf) !tr>r'!J thp pest belovJ the eCOnOrTllc. thn"JICJld (Bpldlr Mid !~(JI'JlrJ, l'JI~t), 1~(JI'Jlrl, r 1985a), but tn datp thpn' dU' no rjf'flnltlvr' rr'·,ult • rdr,JtJld hr·f·f Ir·'. hd'/(' shawn potentlal as predators of th!' dlffr'rpnt (,taqr~', (Jf th!, In'.r'r! (I~dlrtr!'. et a7 ,1990). Stralns of fungdl pathofjen:. of /1otarhl/lum anl>O!J/ld(' ,HI'J • Beauvena basslana agalnst the larva(! haVI! bepn i',olatr,rj (lr·Mlr. MIr] {ul(~, 1988) wlth efflcary studles CQntlnulng. rI ·~turJy wa', stdrtprj ln the fall of 1990 ta evaluate stralns of the • entomopdthogenlc bacterlum, BacIllus thurlng7ens7s, with actlvity agalnst pest lolf.!opt{!ra, as mlcroblal agf~nts agalnst the carrot weevll Ta be (>fff!ctlV~. th(; bartrnum must be:? inqested by the lnsect for protoxln ta dIS '.0 1v f> tHl cl t rd n " f 0 rm i n tua n a c t 1 ve t 0 X 1 n b y me ans 0 f l 1 mIt e d p r () t pol Y', l', b Y t h(> 1 n ') pc t 9 IJ t pro te a ses (l ut hY e t a 7 , 1982 ) The l a r val r;taqer; occur wlthln the carrot root maklng Il dlfflcult to incorporate the bdctl'r1Um lnto thf' Hl',PCt's fp0(hn9 habItat Therefore, control should be dlfllPd at the leaf f(l('dlnq ',tagf's (adult) of the lnsect. Ihp oXprnmf)nts ln ttllS stlldy wf~rL dr:Slgned ta· 1) fxamlne the pathot:JPniclty of seven strains of Bacillus thurlnglcns/S dlrectpcj a~j(llnst the adlJlt stage of the carrot weevil. ?) 1 Xamlnf> thp effl'( t of Intoxlc.ated survlvors upon discontinuing their (lxpoc,urp to thp Insect]( idal crystals. 3) [Jptprrnlnp ttw mldqut pH of the aduH carrot weevil and its t' pla t 1 0 Tl ') h 1 Il t () Ct' ys t d 1 s () 1u b 1 1 lZ a t Ion. 4 ) 1 xarnlm' th(' InvolvempTlt of two kry steps in the mode of action of ttw cl endotO)lln, ),('. c.rystal solubilization in vitro, and • proteolyt lC fH'(l(('SSlng with gut JU1ce extract. lh1~, thesl" lS wntten ln a manuscript format and the following ('X( l'rp t frolll (iuJ 9.1"' J Jlli~_~.9nCerr:Llllg_~i}f~~~_ÏDilla rat ion, s ta tes the (01)(1111011\ undPt' whlC..h the thesls must be wrltten. " Tli (' (ù n d 1 d ,lt (' h il ~, t li ('op t 1 0 n , su b Je c t t 0 the a p pro val 0 f the d('pdt tllll'TlI, of IIlCluÙJrllj d', part of the thesls the text of an original pdpL']', or papers, ,)lIl tab 1e for subnll ss 1 on to l earned journal s for pullllCiltlOli ln thls cac,e Ulf' th(~SlS must still cOTlforrn ta a11 other 1 f' ~,ulflrll'l1t dddil tll JllOl~ a clellr and preuse Judgemrnt to be made of the lillportallll' dnd Ol'lqlflùllty of the research reportt:'d. Abstract, full lTlt"lldlldlllll dlld l()ll(lu~lOn must tH' lnlluded, and where more than one IlIdIlU\Cl'lpt dppPdrs, cOI1Tlectlng texts and cornrnon abstrè.cts, introduction • 2 and conclusions are required. A mere collectllln of !1lùllUStTlph 1" rhl! acceptable: nor can repr1nts of publ lshed papers be arreptect. • While the 1ncluslOn of manuscnpts co-adhered by the Cllllll1datl' ;\Ild o the r sis no t pro h l b l te d bY Mc G1 11. the Ca n d i da t f' 1 S WLi l' n l' li t 0 111.\ 1<. l \ ,1 Il explicit statement on who contributed to such work and to what l'xtl'nt. ,1I1d Supervisor's and others wlll have to bear wltness to thp aCL'lIl',llV of \lItt! clalms befon' the Oral Commlttee. It should also Ill' Ilotl'd th,lt thl' td\I<. llt the External Examiner lS made more dlfficult 111 such Cél~e~. ,111d Il 1\ 111 the Cand1date's interest to rnake aulhonh1p re':>poll\1bi 1 ri Il'', fll'l'll'lIlv clear." There are two Chapters presented dS papers for 5ul)(111\\1011 to lhl' Canadlan Entomolog1st (Chapter 2) and Journal of Inverlpbrale P.tll101o • 3 • CHAPTER 1 LITERATURE REVIEW • • 4 • THE CARROT WEEVIL Taxonomy and dIstribution The carrot weevll, Listronotus orcgonensls (Il' lord!'). (Coleoptera:Curculionidae) was first identifi0d in 1860 d~ ',~tIOdl'n.'~ oregonensls (Le Conte) and was subscqllcntly l'f.'nlHllt'd ',st"0I10tU<, oregonens7s in 1876 (Henderson, 1939). Othcr synonyrn~, lISPc! lo dpllotp thl~ species lnclude: L7stronotus latlusculus (le Conte) Llstronotus lmprcsslfrons (Le Conto) Llstronotu5 rudlpcnnls (Blatrhley). The carrot we0vll 1 s ndt i ve ta North Aml'r 1 ca PX tend 1 Il!) f rom !)UPlll'l ta Flanda and northwest ta Manltaha (Buchanan, 1932) . Pest status • The first record of carrot fJeevil InjUries wa', ln 190( III VlY'qlnrd on parsley W'th :)ubsequent ')!10radlC report'" from III !flOr'" LOlll1t't IC ul, Np\'J York, and Washington D.C. on crops of parslfly cHHl Cdrrot·, (PPPllf't, 194?), In 1925, 50 tù 90'1'0 of the carrot crop Wei', lflJ\lY'pd !fI Jilin/li', (1 hdndlPt, 1926) and the carr'nt wecvll redchfl(j w'c;t :,Lltll') Irl JO'tJd (ILltt'!'" 1<)7G) Severe outbreaks on celery and (M'fot:, ()((urrp(j ln ~J(lW ,)PY',Py ln 19% (Pepper and Hagmann, 1938), and on cdrrols HI Md')',adlll','.pt t" IT! 19~)G (Whitcomb, 1965). Presently, the carrot wprVll rpmalw. d pP'.t ln ',OIlIP parts of the northeastern Unltf!d ')tatr', of Arnprlfi! (Crdflll', rt al ,19rr3) 1nOn t a rio, t h f} Cil r r () t'vU) P vil wcl " f 1 r', t ]( j Pn tif 1 (> d il l t h (. t Il r ri () f the c.entury but dld not ac.h)f!ve econornlc lmport.lnc p Urltll 1969 ('J1f'VI'n',Orl, 1976). The flrsl lnvaSlon of the rarrot weevll 'tJd') obsprvprj HI 19S6 Irl the Holland Marshe:; and Bradford (Harc.ourt, 1959) and by 1963, th(' (drrot weevll was beglnnlng lü cau:;e concern (Harcourt, ;')63). Thp flr',t ',lfJrl (JI the carrot weevil ln Quebr::c wac:. ln 1967 HI Stc~. (lotlld(· (f)('Y rrHi , 1971) It galned economlC Importance by 1970 whr!n dama(J(! rangf!rJ bdw(>prl ? Mid 'I('j" • 5 root inJurH~~ of the carrot crop (Perron, 1971). In both provlnces, the (arrot WCf!V11 attac.ks are restricted ta the carrot crop. Ta date, the • (arrot weev 1 l (ont 1 nues to cause prob 1ems to carrot gro\>Jers 1 n bath OntdrlO and QUübec (801vln and Sauriol, 1984; Stevenson, 1985). D~scrlptlon of stag~s Adult Adult wecvils are oblong, black, with cupreous tinged scales (Whltc.omb, 1965). The antennae are clubbed and reddlsh brown ln colour. Ihl' ~tripec, on the thorax and the mottling on the elytra are a result of thl' prosence of a lùyer of dark scales. The elytra are stnated and out Ilned by row; of grey setae. They have well developed wings but are rarply ~een ln fllCJht (Boyce, 1929). The adults vary cons1derablv in size with the males belng the ',lll(t1I('~)t (lable 1) (Martel ct al., 1976). However, the sexual dlmorphism III ',11(' 1~) not an accurdte means of determining sex. Accord1ng to Whltcomb (1965) male', (ôn tJP sl'parated from females by examllling thelr first • vf'rllr'dl atJd0I111nal soqrnent, Uns segment havlng an oblong deprflsslon in m,t!p'. dnd bl!ln9 swollon w1th a small oval depression ln females. fgg Hw ('~J~IS are elliptical and uniform in size (Table 1) (Martel et ,J/.,!97G). [3oth fertllized and unferti1ized eggs are inlt1a1ly 1ight y<,llnw ln (olour with fertil1zed e~Jgs becoming darker wlth age and almost hLld, Just prlot la ec10s10n (BdUdLJln and Boivln, 1985). The posItion of thl' l.1t'vd 1'> pa'>lly dl~,c(,t'nlbl(> HI the flnal stages of development. The l.\rVd ll~e~ lb l1land1bles ta puncture the egg membrane and leave the egg. L lrva lhp larvae are Sllghtly curved, legless and creamy white in colour vprl11lfonn larval instan (Harris, 1926; Martel et a7., 1976). Larval • 6 • Table 1. Dlmensions of adult and egg stages of the carrût weevil, Llstronotus orcgonen~l~ Length (mm) Width (mm) ------Adult male 6.00 ± 0.30 2.20 t 0.10 female 6.50 ± 0.30 2.40 ± 0.10 Egg 0.80 ± O. la 0.50 a 03 • (Summary data from Martel ct al., 1976). • with a sclerotlzed amber head c3psule (Whitcomb, 1965). There are four lnstars that tari b0 dlfferentlated according to head-capsule width which • exh1blts exponpnt lai qrowth (Table 2) (Martel et a7., 1976). The mouthpdrt'• .1n: flJnc.t lon.11 throughout the instars and the srl1racles are promlnent. A (on~l~tent pdttern of setae lS present on the head and body ')(~qments (Whl tcomb, 1965). Prepupa and pupa Thp prppupa 15 a nonfeeding stage of the fourth lnstar (Martel et al., 1976) and meastJres 7 mm long and 2 mm wi de (Whitcomb, 1965). The larvae t0d5e ta feed and pnter the prepupal stage without moultlng (Martel et a7., 1976) lhp pUpdP are pxardte, creamy white, 5-7 mm long, and have a typical turtullOn1(j form. fhp head, thorax, legs, and abdominal segments are Ilned wIlll stiff sp1nr<, (WI-j1tcomb, 1965). If dlsturbed, the pupae flex thelr bodlf"J (Peppf'r, 1942). • 1 ife cycle Although there ilre 2-3 generations of the carrot weevil in the UnIted States of AmerIca per year, this insect is u5ually unlvoltine in Canadù (Wh 1 tromb, 1965, Perron, 1971; Stevenson, 1976). Adult weevil 5 ov('rwlntpr both ln tlli' flp1d borders and in incompletely harvested carrot fl!'1d .. (Cldflll" and C011111'), 1986, Pepper. 1942; Stevenson. 1976). The dpvl'Ioprnpnt of thr' CdlTot wcev11 lS temperature-dependent (Martel et a7., 1976, ~ II11lllWt dnd [)Jvpnpot t, 1981). 1h(' overW1 ntered adul ts have an oblHjdlory pl'(,llVlpO~\JtlOJl pl'rlod (Whltcornb, 1965) whlch avcrages 17 days dt 21 0 L drHl la ddY\ clt ?loC (Martel et al., 1976). Pepper (1942) reported that Ill'wly pf11prql'd fl'r11J1c', maled pnor lo overwlntenng but, the usual tn'Ild 1') fot' OVP1'Wllltl'rL'd adults to mate ln Srnng (Whitcomb, 1965). In Ldllada, OV1po<'ltlOn starts ln carly May (Stevenson, 1985, B01vln, 1985a). !\tconlllltj to Bdudoln and B01v1n (1985), total fecundity 15 neither dffected by the number of matlngs per female nor by the number of females mated by a SIngle m~le. The female chews a cavlty ln the petiole or crown • 8 • Table 2. Head-capsule width of the larval lnstar') of the c~rrot weevil, Listronotus oregonensls Instar Head wldth (mm) ------0.31 l 0.002 1 1 0.44 ± 0.004 1 11 0.65 ± 0.004 IV 0.99 ± 0.004 • (Summary data from Martel, ct al., 1976) . • of thr carrot, df':pOSltc:. 2 - 3 eggs and seals the site with a black f!xudate from her genital segments (Harris, 1926; Pepper, 1942). The daily • number of eggs laid lncreases as the temperature increases from 18.3 oC ,Jnd 29 4 0(, the eggs requlr1ng 12.8 and 4.8 days for ec..loslOn, resp{!ctlvply (Slmonet and [)av~nport, 1981). The first Instar larvae Jrnmf!dlatply start feedlnq ln the root. The larvae ln the petioles p~netrdte the root~ clther by tunnelllng down into them or by leaving the c,tdlk and Invddlng frutTI the SOlI (Stevenson 1981). The average dpvplopmpnt tlme for thp flrst three lnstars lS 13.1 days at 23.9°C (~Imonet and Davcnport 1981). The developmont tlme for fourth-lnstar larvae in the flPld l~> double that in the laboratory takln~ 6.5 days at 21.9°( (Slevpnson, 1976, Slmonel and Davenport, 1981). The fourth-lnstar LI r v deI fi a v p t h (' C arr 0 t ~> and f' n ter the pre p u p cl l ,; t a q e i n the s () i 1 (r (~P Pe r , 194?) Thf'y cOn':,trtlrt an {~ddhen cell by bcnrhng thelf oodlE>" back and forth rapldly, ('ompdCt lnq ttlf> surroundlng '..011 (Harris 1926, r1ute1 et dl., 1976). At thr· ~,drrIP temperature, pupde require an average of 6.8 d,IY', to drvelop (~Inlfllll't dnd Oc1venport, 1981). Teneral adults remaln ln t hl' 1 r ellrthen ce 11~, fOl cl ';hort t l me, unergt' from the ground and feed on folld dill (Pl!PPl't' dlld HdqrndrHl, 1938). Ldboratory experlments of Slmonet and Ildvpnport (1981) p:,Llbll,)~ll'd thJt at 18.3°( (In edrly Spnng) ancJ 23.9°C 'for (SUlIl1lll'r), ri full cycle frolll egq to adult would requlre a minimum of 3e.:? d,lY<'. !\ddlWI 17 days clS a Illlnimum preovlposltlon penod brings the tot,Jl numbpr of cJays tü complete a slng1e generation to 55.2. Dd111a~le to the crop 1 S caused by both the adul ts and 1arvae. tlowt'vl'r, thp lnJUrleS caused by the 1arvae are of a more serious nature. Adults The adult carrot weevil can feed on all exposed parts of the carrot, • 10 the feeding marks appeanng on the fol1age, the petlOles Jnd ttH' (t'own. Similarly, OVlpos1tlon traces are left on exposed req10ns of the plant. • Such damage lS not of major importance because it does Ilot tllfl(lpl' thl' development and marketability of the plant. Larvae The larvae eat thelf way lnto the upper one thlrd of ttH' carrot root result1ng in galleries llned with damp sticky frass (Pppper. 1942). lh('1'1' are usually more than one larva per carrot dnd thp~e fl'ed 111 thl' root form1ng dark zig-zag tunnels which orten JOln toqethpr to produu' cl brode! lesion (Anan, 1977) It 15 bel jpved that ln the pasto the carrot ru')l f ly. P~IIt1 rO~,Jll, was mlsldentlfled ae; the a confûslOn was overcome wh en il carne to be knowll thdt (dITot tll',t flIP', form tunnele; in the lowor lwo-thlrd spctlon<; of th!' CinTot l'oot ,Ill!! Il,IV!' smaller dimpnslons th an thO~fl of thl? carrot Wf'f'VII ("ltpvpn',oll, 198\) When left unconlrolled, tlw Cdrrot weevIl (,ln (Lllnd the Que bec car rot cr 0 p ( 80 l VII l , 1985 a ) ') 1 n «(' 1h f' Il cl C k 1 nq d Il ri (d nIl Hl< J • industnes can toleratp no mon: than 5'Yo root InJllry (PrrroT1, \g/l), It l', eVldent that sorne pest cont rol l S usually nece<;<~c\ry. Types or cont ra l Ch' In the past, L. oregoncnsis has bcr~n controlled with IW,P( t 1< P]I", (Martel et al., 1975). Sy:.temic insecticidc!', are reqlJlrflrj to r(ldCh thl' egg5 and the larvae, while the adulte, dre tdrqr.tr:d wllh follM applications of contact lnsecticldes. Oryaflochlorlnr!,:; (hl'ptd(hloY ,Hld aldrln) (Whltcomb, 1965), and ar~enlc.dlc, (calcll.Jm ar~f!ndt(·) (l'l'ppl'r, 1942) have bel;n used. The· orgdrloc..hlorlne-:; Itlerl~ must dff'ctlVl'ly dppllprj as granules ln the 5011 follo\'lC:d hy folldfJç! appllf.atIOn',. HOItIP,/I!Y, thr . tOX1CIty of the more persIstent lnsectl(.ldes on the carrot root ha', lPrj 1.0 • Il thr: uc,{' of follar dpplicatlons agalnst the overwintered adults pnor to oVlpositlon a', thr; only means of chem1cal control (Brodeur, 1985; BOlvln, P • 1985a). Presently, phosmet (Imldan ) lS registered fOI- this purpose in OU0ber at the rate of 2.25 Kg/ha tWlce seasonally (CPVQ, 1987). Cultural control Annual crop rotatIon effectively controls the carrot weevil (Anon, 1977). Slnce the adulte; rarely fly , the presence of this insect in the fIeld 1:; a \'/drnlng for an infestatlOn the followlng year if the farmer dtt(!mpts to yrow carrots thf.:'re once agaln (Stevenson, 1981). However, df'tectlon of the adults rf'quires efficl(.'nt monltonng whlch, ln turn, is • :rnr: con~urnlnrJ dnd costly MonItorIng progrdms for thlS pest lnsecl have h(>~IIHl ITI Mldl1qdrl (Grdf1us et al., 1983), Ontario (Stevenson, 1985), and [)uebpc (Bolvln dnd Sauna1, 1984). As an alternat Ive ta monItoring insect popllLJtlOTl d('nsltlP~, d trap crop surrounding a carrot fIeld was spnyed \·,lth JrI',I'CtIClc!P3 l'IllICh successfully redured the level of carrot weevil Irdp',ldlICHI Oll tht: Cclll'ot (\>Hlltcomb, J965) /\nothl't lulturdl practlcP used ta escape severe ddmage by the can-ot • v/l'l'VII IS to dpldY the date of plant1n9 (Bo1v1n, 1985a, Stevenson, 1976; (hdr1!llpl'. 1926). /\ slgniflcant lnVl'r~e relatlOn exists between the date of 'owlnq cHHI ttH' nllmtwr of e ldllOI l'IP(lvII cldlllclllL: WJ~, ellm1nated when carrots were sown after 400-450 llil However. thlS pract1ce i5 not usually !O]]()\'Il'd. "1 III l' It tenJ~ to reducp the yleld of the crop. Hl%glcal control /\lth()ll~lh tnologlcal control 15 another possibility to mlnimize d,\r1l.Hlt' (dW'l'd [ly th!' can'ot \'/eev1l, surpnsingly little information lS t'llt'lt't! ln LIll' llterdtllre lhr mYIlI,lrld WJSp Anaphes sordldatus (Hymenoptera) 1 S an egg l'dl,I\ltOld llj L cn:90llensls (Collln and Graflus, 1986a,b,c) parasitiz1ng ~lL tlf ldlTllt II/l'l'VII eggs ln Qllebec (Boiv1n, 1985b). Allotus curcullonlS, • 12 c a braconid wasp, was found to paras1tlze 2 ., of the carrot WN'V11 Llrvdt' ln Massachusetts (Whitcomb. 1965). On the other hand, I-H'pdatory cdt'Jtnd • beetles :Coleoptera: Carabldae). Wh1Ch an' abundant in carrot fll'ld" ot southwestern Quebec have been shown to consume dlffel'ent ~LI~lt''' of ttH' insect (Ba1nes et al .. 1990). However. 11kE' most pdl'a<;ltl'~ dnd pn'ddtlln. they wou1d reqIJlt'f~ slr'p1t' Jnd cheap f1lJSS 1'(,dl' 11)(1 tl'chllJ(llJt'~, III tw effective. and these have yet to he des1gncd. Laboratory studlrs on the sU"L'eptltlllltv of tt1l' can'ot wl'l'v11 "Ll~ll'" to thl'ee entomogenous Ill'matodes 5tc/ncrncma carpocapsac [S. f('/t /dL' (Po1nar, 1989)1. 5. fclt/ae l=5. blblOI1I'i (Poinùr. 198Y)I. selected vIrulent str,Iln'; of Nctarh771um an/sopllac dnd (?cauvcrla hd~)lanrJ by me ans C' fla bol' a t 0 r y b 10 a " ~ a VS el ~l a 1n c; t t hp 1cl r val '> t cl CI (\ '> 0 f t h fi ( A new c.ûntnbutlon to the fluebec Intf:9ratrd Pl",t Melna!j!'ntpnt l'tfJqtdlII for the control of the carrot weev11 would hr the u<,(! of ',trdlrt', (Jf dll entomopathogenlc bactel'luill. [jac/llus thUrlng7cn~ls, thd! 11<1\11' ',h()WIi activ1ty aga1nst Lo1f'Jptcran srWllt:S. BACILLUS THURINGIENSIS Historica7 background Bac/llus thunngUJns/5 wa: l::.olatr:rJ at trIP turn of thl> rr:rdlJYj hl a Japanese bacterlolog1st, S. Ish1wata (Ishll'/ata. 1901). rhr: p;jtho'J('n If/d', ls01atEd from the silkworm, Bombyx mOrl, dnd wa" ndm(~d )(Jtt()Vlrt \tJhlfh • 13 mpans " suddç:n death bacillus " (Ishiwata, 1905). At the time, thlS OfljdnlSm ~Ja', cow,ldpred a threat to a very important lndustry, silk • production (luthy ct al., 1982) It was not until decades later that its potentlal U',(> for IW,cet c.ontrol wa:, r"eallzed. lhC' ',econd 1',olatlOn wa') mauf~ by E. Berllner ln Thunngla, Germany from di-:'f~c1',erj larviH' of thp Mr.rjlterrilnean flour moth, Anagdsta kuehnlella, (Zpller) dnd lt ~Ji1r, ndrnpd f3ac1l1us thunngiens7s (Berllner, 1911). B(>rllner rjp~u'lb(>d lt ilnd ShOWf;d that lt fulfilled Koch's postulates, thus valldilt Irtq hl', rldllllnfJ (JI th!; l',olate (Luthy et a7., 1982). Wlthln the prp',I:nt L1XOrlfJnly, thp ',tt'dlrl rl(!'.Lrlbf~d by Berliner has bpen designated as ',IJh'.p('( IP', thunngu'nsls (lilthv C't al., 1982). BctW(lPrt 1920 thur1ng/(~nsls W('rl' Ilfldf>rtdkl'T1 l'Jlth vanous degrees of success. Sporeine, thf' f,r-~,t (OnirTIPf(lill prllduc1 of [J. thurrnglcnsls was manufactured ln Irdlltl' around 1938 (Idnlbl'rt dnu Peferoen, 1992). In the 1950s. Hannay (1953) dl~(Ov('r('d lh(~ (l(tlve lngreUH?nt of B. thunngiensls and in 1956, I\rHII/) (1956) provcd thdt the Hl~ectlcldal actlvlty was located ln the pdt'd,>pordl ITlC llJjlOn [)urlnq lhl' 1960~., d nUlllber of lndustnal formulations of B. • tlJU/'lnglCf1\/S wel'l' produlpo ln the United States of America, the COlIllllOTlWpalth of Indf~ppnrJent States (former Soviet Union) ,France, and (,Plllldny (ldrnht>rt dlld Pefproen. 1992). Bù')ed on B. thunngiensis \Uil')Pf'l If". th'u'/f](]/ens/:" the ln)cctlcldes had luw activity and could not tOlllpl'tp wlth chrmlCcll lnsectlcldes ln pithet~ efficacy or co st (Becgle and YdllldlllOto, 199?). ln 1962, [ Kllt'~lùK lsolated a new subspecles of B. thunnglens75 fl'ollll1. kuehr71clla larvar ln France and ln 1970, Oulmage lsolated the same '.llt1\p('( H') (Wtlllh ttH:y narnod kurstakl) from thf> plnk bollworm. Pectlf1Ophorù gO"iHplc77a. lhp lsolate proved to be more potent than the o.,lI!l"[H'lll') thunng/cns/:, and wùr; con~netltlv(, wlth ch0mlcal lnsectlcldes (11Ulllltlq(', 1970) l'I't)~l'lltly. sl'vpral nJllllOn KlloCjrams of kurstak7·based produdo., diT m,lflllftlCtuno Jnnudllv ln th(' United States of Amenca wlth Il<;dlll' l'l)ljl')ll't'l)u fOI' approxlrT1Jtelv 30 crops Jnd agalnst more than 90 pest l!l,P\ t .... Wlll'ldlvldp (flPl'qll' dnc1 Yamamoto, 1992) . Untll tht~ 1970~, rnost of thE' descnbed B. thuringiensis strains had • 14 ------ demonstrated activity against lepidopteran lnsects. In Israel ln 1976. Goldberg and Margalit (1977) lsolated a B. thurJngicnsls with mo~qlllto and • blackfly larvlcidal actlvlty. It was nalllcd SUbSPl'CH'S IsraclcllslS (dl' Barjac, 1978) and had an outstandlng level of actlvity and '>pppd of klll. The most reccnt B. thunnglensls lsolatp to hav(' (llJl1llll'l'llJI pl'Onll\l' is subspecles tenebrioll1s. In 1982, KrH'q ct al lsoLltpd from }('ncbl'lo mo7itor a new stralll of B. thunnglcnsls path091'nH ,lq,lln\t Cl't~tJln coleopteran lnsects and namrd lt B thunngicnsl~ "lIb'.p t('l)('hnonlc, (Krieg et a7., 1983) In Sdn D,ego, ln 1986. cl ~tralll of [) th/Jl'1n91('n~l~ with simllar actlvity was descl'lbed by Hprrnstadt ct a7 "tnd Wd'. tl'I('n'l'd to as B. thunnglcnsls sub',p. Sdl) dlCgO lIowpvpr, 1 t W,I" \ub''!'qllf'nt Iy demonstrated thdt both stralW. werp Hlf'ntl(ùl (Kri('~J ct al., 1907<1). The sus cep t 1 III 1 1 ~ Y of '> (' Vf' r d 1 ( 0 1 f' 0 Pt (' r d n sil P ( l (' " III ( 1 Il fi 1 nIl economlc.ally Important pests (e~l., Colorado polalll twpllp Icptlf70tana decemllncata, and the LoUan bull Wl~t~Vll, Anthonomu~ qlafJ(J,',) to fi thuringlcnsl:' ~)uhsr tcncbl'lonlS (Hf'rrw,lddt ct al. [gOG) hù" atll'd(tpd attentIon to the SUbSP(·C1C::' HI terrn:. of Il', Pftp(tlvl'r]!',,'. d'. ,1 bl()(Ontrol agent (Herrnslddt ct JI., 198G: Hlcthnltllll't dlHj lanqf'nhru(h, 19H9, J prro and Gelerntp\" 1989, h'hndf'r ilnd G\'lptnlet-, 1989), Il', t)l()dlf'rlll'.try • (Bernhard, 1986, KrJP(j ct dl., 1987b; LI ct al., 1988; Ctlrroll pt al., 1989); as well as lt~. molcLular blOlo Most studlcj of the rfff'ctlvrnp',c, of H. thurtnqlcn~/~ subsp.tenebnonls haVf becn for U1p c.ontrol of 1. dCCt.'m/lncatrJ, thf> fl1O',t destrllctlve Pf!:t of p(JLlt(J(l~ ln lIorthf'd',tPJrl Unltprj )tdlp', of !\rI\f>Y'lCd (Ferro and Gplerntcr, 1989). 80th Lltj()rcltory dnd f J('ld Ipc,t', Itld I( dlpd that the pdthogc'fj ('fff'ctlvply (ontrollf'd thf> IdY'Vdl POlluldt lOTI of ttll', pest on leavc:J It Wtl" û1:.o not('(j thtlt Ulf' toxln l'> bl0deqrddclblJ> no known effect on nf)n-tarq~t or Screenlng of B. thurJnqlenSlj suh',p tencbnonls l ',ol,J!p', for lnsectlcldal actlvlty ,(lveali~d tbdt fdmll1f!', tri th!' ()rd(~r Colpoptera dlffered ln thclr susceptlblllty to the straln (ldblp 3). There are numerou', formulatlOns ot B. thunnglensls sub::,p. tenebrlonls (lable 4) (Heegle and Yamamoto, 1992). • 15 • • • Table 3. Insects evaluated for susceptlbillty to Baci77us thur7ng7ens75 subspecies tenebr7on7s (= subsp. san d7ego). ------==------Order Famll y Species Stages tested ActlVlty Coleoptera Chrysarn;::lidae -Dlabrot7ca A, Ll, L2 + undec 7f71puncta t a -Ha7t7ca tombaSlna A, L2, L3 +++ -Leot/notarsa decem71neata U, L2 +++ -Pyrrha/ta 7uteo7a A, LI, L2, L3 ++++ Curculiondae -Antho~omusgrandis A, L2, L3 +++ -Otlorhynchus su7catu5 L2, L3 ++ Dermestldae -Attagenus un7calar L3 Ptwidae -Glbblum prylloides A Tenebrionidae -TenebrlO molltor U, L2, L3 ++ -Trlbo71um castancum A, L3 Assays agalnst DiabroL Ica Ul7deC7mpunctata, Lept7notarsa decem17neata, and Pyrrhalta luteola were carried out by spraylng spore/crystal preparations, or purlfled crystals on to leaf dlSCS from approprlate hast plants. All other assays were carrled out by lncorporating spore/crystal preparations or purified crystals lnto an approprlated dlet mlxture. Insectlcldal actlvlty was arbltrarl1y classifled from weak (+) ta "/ery strong (++-<-+). A, adults; L. larvae. Assays agalnst Otl0rhynchus su7catus, Attagenus un/ca7or, Tenebrlo mallto, and Trlbo71um castancum were carrled out for 30 days. All other assays were carried out for 7 days. (From Herrnstadt et al .. 1986). • Table 4. Baeil/us thuringiensis subspecies tcnCbt'10171) bù~l\d commercial products. Product Reg i strat i on date Manufactut'cl' M-One 1988 Mycogen M- Trak 1991 Mycogen Trident 1988 Sandoz Agro, Inc. Trident 11 1990 Sandoz Agt'o, III ( . Foil * 1990 Ecogen Di Terra 1991 I\bbott Lù bo r il t () r Il'', Novodor Pend i ng Novo IndlJ)tt'1l'~, • ..... ______• ______• ______• ____ '-3 * Combination of subsp. kurstaki and tcncbrionls (from Beegle and Yamamoto, 1992) . • Noml!nclaturc • B. thunnglcnsls lS a Gram-positive, endospore-formlng bacterlum of thf> farTIlly Bacillaceae. Although it 15 Slmllar to 8ac717us cereus, lt lS ('Jrt',ldered cl ~r.paratr: species largely due to the presence of parasporal cryr,1.dl:, ln U. thunnglcnsls (Lambert and Peferoen, 1992). The need for a method to ,df!nttfy and classlfy B. thunnglens7s 5ubspecles became rl(l(f>j',ary a', dtffr!rPnt IsoLltr;<, were discavAred ta have olfferent actlvlty ',pPftra, and lJ. thunnglcnsl5 bl'came lnCrE:dslngly c.ommerclally lmportant (Bf'f'qle dnd y,llTlamoto, 1992). Inltlally, the methad was based on lI1orph()logHal dnd hlochf'1TI1Cal charaftr:'rlstlCS (Hplmpel. 1967; Helnlpel and I\nf/w" 1958, 1960) dl' BdrJJC (1981) and de BdrJdc and llonnt'fol (1962, I%P, 1973) InttOdlj(pd a clas<,lficatlUrl scherTle bac;ed on serologlcal MldIY',l', of vpqptatlvP (l'Il flagellar (H) antlgens and blOchemtcal (hclrrlftpY'lstlC',. Sl'vC'Y'dl oth(lY' ldrntlflc.atlon and classlflcatlan Illethods vJPf(' dl',() dflVf>loppd, "lJch as esterase analysls of vegetatLe cells (NoY'r1<', 1964), u'y<,tal ~.(>r()lo9Y (KY'ywlenclk and Angus, 1960, Kr'ywieczyk [lt al., 1978, ~mlth, 1987), and slIsceptlblllty of B thunnglens7s ta phall(' (\)01](", ct .JI ,1983) HovlE.'ver, flagpllar serotypHlg became the • IIlpthod of thol((l nldlnly twc.ausr a flagrllar St?t'otyplng SerV1U? was made ,IVcllldhlf> bv ttlP Ir"lllut Pasteur. Present Iy, there are 36 serotypes of B. thurlllglcnsls (Bppqle dnd Yafllamoto, 1992). Hn mOl,t \'P(pnt tJxononllC procedures lIsed lnclude Hlgh Pressure Ilqllid Lhromato tvP(\\' wlthlrl cl ',l'rotype) and plasmld map[nng, and genE' cloning and ',(lqll(If1ClrHj of thL' Cl'ystal toxln (Beegle ùnd Yamamoto, 1992). A new LIXOJlOflllC "y~t(,fll !t,l', bl'l'Jl propased for B thunnglensls crystal genes (( t v) hd)pd on thl' typo') of crystdl protclns produced and the activity ',[1('\ tl'UI11 of th(' ln~('ct lC1(Lll toxlns (Tùblc 5) (Haftc and Whltr:ley, 1989). \lll\{' ttlt' pl'lfllJry lrltPt'l'st ln B. thunnglcnsls 150 in lts abllity to kill P(\\,t ln<'PlL, 11 Sl'erns llkcly that thf> ldentlflcatlOfljclasslficatlon ',dll'fll(l based on structural Slmllarltles and lnsectlcldal spectra of the (lnlod('d protplns wlll replace that based on flagellar serotypes • 18 • Table 5. Host speciflcity of crystal protelns Gene type Host range CRY 1 Lepidupter • ------ Sporulation and crystal production • St. Jllllan et al. (1971) documented the different morphological st.ages from the dormant spore to the vegetative cell of B. thunnglensis. A brlef descrlptlon fo11ows. Thp veqptat lVf! cel 1 s of B. thunng7ensis are roù-shaped (average dlmenslOn of 3 Jjlm x 1.2-1.5j.lm) perltnchously flagellated, and usually o(cur d~, fI1drPf!nt" of tlt least four cells. VegetatIve cells are known to prorJ1HP Wrllpt ')olutJ1p, lrl'oPc..tlcldal toxlns (see sectlon "Types of toxins othpr thMI Ihr· c'.-endotoxHI") Il thurlng/()n',J~ l') cornmonly found ln natural sOlls and ln pnvlronnwnl'. wlth tllfjh donsltH?S of lnsects such as graln storage and HlsPct f(ldrHHj fac 1111 IPC; dnd sencult.ure farms (Lambert and Peferoen, 1997) Thp dorllldnt '.pnrf''' elrE' able to SUrVIVI! unfavourable condItIons over d lonq pr\llOd of tlnll' (tllthy et al, 1982). Thplr reslstancp to heat, lhl'rTIHah, lYTddlat Ion and de .... lccatlon 1" attnbutrd to the dphyrirdtlon of thf' <'porp cytopldsm dlJrlllCj sporulatlon. In nature, the sporp<, undergo dCjl'lnq, d ~>low proC0<,') of actlvatlOn WhlCh causes thf' breakage of dl')ulfld{\ llnkaqu" of sporl' (Ddt cystIne thus causlng il chanqe ln the • tprllary ')tructurp of th!' prot.elns WhlCh 15 orl~nnally responslblr for the d,lrmant statp of thr spores. The lnltlatlOn of germInatIon is an lrrpvcrsiblf' proces~; (Lulhy et a7., 1982) A senes of degradative rl'actlow, occur dunng germlnatlOn and are followed by a period of blo<;ynthl'Iic actlvltles necessary for the outgrowth of the vegetatlVe CP 11 . lhp rXhalJstlOn of tnp mpdlum especlally of thelr source of C, N, and P lndllCP'> thE' InItIatIOn of sporulatlOn (Lllthy et al., 1982). The sporulatlon pracess 15 suhdlVlded lnto seven stages (FIg. 1). During stagp 1, an aXldl chromatln fIlament IS former!. ln stage II, the chroma tIn fIl ampnt c;epara t('~ 1 nto two chromosome <; and a subpo la r forespore .... ('ptUllI lS fonl1pd d[llllllltln~l the slte of the future spore. Durlng stage Ill, l))O~j!'rH'SlS of a crystal beglns. The crystal is a prot€~lnaCeous pdtdspot'ùl lncluSlon known as the b-pndataXlTl and is lethal ta specifie ~lrllLJpS of Insecte,. Il accounts for 20-30% of the total proteln of the spordllgllull (Aronson et al., 1986). Stage III 1$ also morpholugically • 20 • Figure 1. Diagrammatic scheme of sporulatlon in B. thuringlensis. Abbrev;at;ons: M, mesosomej CW, cell wall; PM, plasm.l mpmbrane; AF, axial fllamentj FS, fon'spore sl'ptum: If, incipient forespore; 01, ovoid inclusion; PC, p lame 11 ar spore COdt; C. cortex; UC, undercoat; OC. out<'Y' ~pore coat; S. mature spore in an unly~ed sporanglllill (from IdU<;t dncl Bu 11 a, 1982) . • • 21 • • • ------,. characteri zed by the format i on of a free forespore with an i nncr and out PI' membrane. Tl1e cortex and the pnmordial cell wall are formed bt'twl'ell ttlt' • ;nner and outer membranes dUl'1ng stage IV. ln tht:> cour-';l~ of <;t<1(1(' V, tIlt' l ayers of the sporecoat develop and thf' exospon um wh 1 dl <;lllTOU11lb t hl' spore 15 completed. OUrlng \taqr VI. the spoY'e mùtUt'tltlOll PIOlt'l'd, ,lIld 1' completed dunng staql' VII At Uns '-,ta~Jl' lytlC Pl1lynH", n'l(, crystal formatIon hdVl' lll'en dl~ClJss('d 111 dPlall 111 r(lV1PW~, pll''-,l'ntt'd by Ribler and Lecadet (1973). and Brchtel dnd Bulla (1976). The pn'domlnent crystals of most Il thunngll"Yn!>l<' \lI!l"IH'lIP" \() LB' identifled are compnspd of 130-140 klll)(Lllton polyppptHIP'. (!\nHl\OIl ct a7., 1986). 1hore arp, howrvpr, dlffp)'rnl(", III 1l11111!lPI', '.b'II)!', dnd compositIOn of thp\p pdraspordl lmlu\IOW, Whll\' LIli' nUllltH')'" l,Ill r.lfllJl' from 1-5 crystdls/ bdctpflal cpll, thl' \hllpf' th,lt thpy (MI ,1'.'.llnll' 1', blpyramldal, cubOld, ovoHi, or amorphouc, (!\ron~on ct al., 19Bb) Types of toxins other than the o-endotoxin • a-exotoxin Also identlfled as possibly lecithlna<;e C (pho<..phollpa'-.p L), Ir exotoxln 1S a heat-lablle protf~in that 15 pruduc.ed hy vPcjPtat IVP cpll'. (Toumanoff, 1954). Howevf1r, Knclj (1971b) and IVIn',klpnp (1978) determlned thdt thl c, toxln was not li lpcllhlnrl',~~. n (lXO!OXlfi W,J'. '.hown to be tOXIC to the dlarnond-bdck moth, Plutclla xylostc/la Il l'. dl'.!) tOXl< ta mlce per os (Kncg, 1971a), hf!nre thp ~.ynonyrn "rnolJ',p f,Htor" Thl' presence of the toxln l~, only demonstrdblc' by blod',say Mid (Mlrlol bp readlly Isolated or pUflfled (Kneg and Ly')pnko, 1979) Il', ',I/f' 1,,45 50 kllodaltons (Kneg, 1986). B-exotoxin The heat stable l3-exotox1n was dlscovered by McConnell and f{Hhard', (1959). It lS also known as a Fly factor, a heat-'ltahle toxln, cl thermostable toxin, and thunnglensln (Beegle and Yamamoto, 19')2). It l', • 23 produced by vegetatlve cells as a water-soluble nucleotide analog of adenlne, rIbose, glucose, and allanc acid wlth a phosphate group (Farkas • et al., 1969). Thp B-exotoxlns produced by dlfferenl subspecles of B. thunnglcnsls ue not dll ldentleal (Mohd-Salleh et al., 1980; Glngnch et al., 1992'1, 1992b). lypf~ II B-exotoxln was found to be more speelflc than tnw 1 and 1'J very actIve agalnst L. decemllneata (LevInson et al., 1990). Iyp(' 1 l3-pxotox 1 fl hd r, a very wlde hast range and aets by lnhibltlnq thr [)NI\-depr~ndent RNA polynerdse (Sebesta and Horska, 1970). In thp Commonwndlth of Indrprdent States, commercial products are used effpctlvely (HJdlnc:;t r>[H!Clr:>S of red mites (Aearl), larvae of house flies (])lptprd, Mu',( ldde) and blowflles (Dlptera, Call iphondae) (Beegle and ydflldrno to, 1992) h-endotoxln The parasporal crystal<; (ù-endotoxins) are divided ;nto three pathotypes aecording to their actlvlty spectra (Krieg et al., 1983): Pathotypp A' The crystals are actlve agalnst lepldopteran larvap dnd arp u"uJlly blpyramldal. The crystal lS composed of 230 • kllodalton dlmers (Holmps and Monrof:, 1965) WhlCh, under alka11ne conditIon" dlSS0clate lnto 135 kOa protoxlns that become taxie on1y after pdrtlal clIlymatlL dl~lL\st,on. YJmamoto and M(Laughlln (1981) isolated from B. thunngll:nsl5 subsp kurstakT a 65 kDa proteln different from the 135 'kOd protpln It was termrd P2 and was found ta be taxie ta both Lrpldoptrran\ and Olpterans Pathotypp B' The cryc;ta1s are hlghly active against mosquito (Lullcldar) Jnd blacUly (SlInullldae) larvae and are presently found in subsp. Israelens15 and a dlpteran-actlve sub~p. morrisoni isolate (Beegle and Yamamoto, 199?) Severa 1 1 rregul arly shaped erysta 1 sare produced per ~poran~l1um. Thc,>e crysLll S drr composed of three groups of proteins: 128- 135 kDa. 72-78 kDa, and 27 kDd Although the latter has little Insectlcldal dctlVlty, lt may have synergistlc action with the former tOXlfl protelns (Wu and Chang, 1985). - Pathotype C: These crystals are actlve against coleopterans and dre located Hl subsp. tenebnonis. They are unique in shape and • 24 protein characteristlcs ln that the crystal 1$ squarp and flilt, and 1~ made up of 67 kOa pt-otCln molecul es (~krherson ct JI.. 1988). Unll Kt' • other B. thur7ng)(~nS7S subspecies, tCf)CbnOnl5 crystals do not hdVl' disulflde bonds (Bet-nhard, 1986). ln additlllll, unllkl' otlll'l' lry"Lll types, NilBr- or alkali-dlssolved crystals rccrystall1ze wllPn NaBl' IS removed or the pH lS lowered (Beegle and Yamamoto, 1992). Mode of action The mode of aet l on of the ô-endotoxln 1 spart 1 dlly aU nbut t'd to 1 t ... three-dimentional structure. The domalns of a tYr>ical pdthotypl' fi. pl'OtOXltl derived from B. thUrTnglens75 IS 1I1ustratf~d Hl f-'~lun' 2. 1111' pl ()t(lXlrl 1', cleaved by inseet mld~Jul pt'otcùses rpl!'il',lnq thr> C teY'f1l1ndl ·,tlll fragment. The remalnlr1 c conslsts "f at least 2 dOrnLllrl ,. the toXIC Jlld u](~ (('11 hlndlllq dOIIl,lIl1·,. There are five ronset'vrrJ n.'Cjl0n<, wlthlfl thp dctlVP fldQlllPrlt. 1111' tOXll domaln conlaln:; hydrophobie refjlons (~lJlld dd)he~,) ,Hld thl' pllt dl IV!' 1011 pore formerl by UIP (xy toxln l', dPrJvpd from ",hpli\p', found Wlthlfl thf' toxln domaln. Thr: U']]-tlln tenebnonlS tox1n called CRY IlIA ThIS loxln 1:' nladp of 644 drlllflO residues whlch are dlv1deu lnlo three dorndln',: l, 1 l, dnd 111 !lllliid 1 Il 1 1S probably 1nvolved ln pore formatIon UOnlùlrl II 1 c, 1tH' rp( Pp 1 rH tJ 1nrl1rI(! domdln. I\nd domdlll III IS thouqht ln prolpct th!' 0 t h r! 1 ri () III d 111', f r r)[11 protelnasp dlClp.stlOn. The dr>termlnatloll of thp ',tru(l11rf' flldkf<', It possIble to determlne the 1nvolvpment of edch amlno ae Id 1!".lIJIlP III thl' 1 nsec l ' C HJ alac t 1VIL Y ln general, the crystal l', an agqnqùtp of il rpl,)t Ivply l.nrlf' prot2lrl suburllt that can h{; dlSSollaterj unrjPY' ',PH !fIC fondit lori', (Idll',t dll'I Bulla, 1982). It 15 lw,olubh: HI walf;r dnd u',llrllly dl',',rJlv"', Imr!l'r alkaln,e condltlons of the Hl',Pct glJt tn rplpi}"p thr: protf'lrl ',lJtJIJrI!t·, (Carroll et al., 1989) r~any nf thp lJrqpr rrystal', dr p hr·ld lol )f,th r,y hy 1nterm';lecular dlsulflde bond', vJhlCh r(!(~Ulr(> vf!ry hlqh pH fH rr,'ll]( In1j cond1tlûf1S to lnltiate dissnlutlOn of thf! c..ryc;tal', Ollc!n:r',rHI, 1':H~O) Ihr· • 25 • F lCjlJrp '2. Domalns of actlvated Bac/71us thuringiensis toxin (from Gill et al., 1992) . • • 26 • N c ACTIVE FRAGMENT STRUCTURAL FRAGt-1ENT protease activation A B C ,u • 1 2 3 4 ~ • TOXIC DOMAIN CELL BINDING DOMAIN • lry~tdl protelns of B. thurlnglensls subsp. tenebrlonis are smaller, and are rendpred Insoluble by salt brIdges (Asp 142-Asp 165, Glu 223·Lys 293) • (Il et al., 1991) !hrlt break dnwn ln concentrated salt solutlOns (t~dBr) ln vItro (Bernhard, 1986) It has been rpported that some lnsects hdVf> il low ',usccptlblilty to speciflc crystal protelns due ta the Indflllent ',olublll1rJtlon of the crystals vlhich, once ~JlublllZed ln vTtro, '.lqrlJflldntly pnhdncp the: tOXE actIVity (JaqlJet et al., 1987). Onet: 'jOlllbl11/f>d, thp subunlts of most crystalr; of pathotype A are prot PO 1yl 1Cd II Y convprt rd 1 nto an aet 1 ve fragm(lnt t F 19lJre 2) by the ml dgut prot(~d'>f>!, [hr; '017e of thp srn,,::'lst active fragrTlpnt, the protease Y'f",i',tdnl UH(', VdrlPS l'ilth the B. thunnglf'nS7S straln (Gill et al., 199?) (l.g. only llmJt(.!cJ proteolysls oeclln al the N-tenmnal fol' CRY 1 protoxln', Whllf> dpproYlmdt(>ly 500 amlno aCld resldues are r(.!movrd from the (t(!Y'IllHldl) In contra';t, CRY II and III protelns appeal' to be naturally trurl(dtpd <..Incl do not Undl'r~lO protpasp-medlat(;d C-ternllnal cleavage. In t.H l, pvpn d :'l11d11 (·t0rmHldl truncat10n of (.!10ven am1no aClCJs may result 111,\ 10',<. of dCtlVJtV Irl CHY lIA (Wldner and Whlteley, 1989) . Thp HlspctlClddl sprllflclty of the toxins lS due to ~llgh-affinity 11IndlrHj lÜ '-.pOCdlC 11Irmhl'an(' l'Cleptors (11 ct al., 1991). The toxin has • hl'(l1l ',hown lu blnd ',ppcdlcJlly to thp brush border membrane vPslcles of tll(' II1ld'llll pplthplllJnI (Clll ct al, 1992) The crystal toxlns requlre a '.pPL!fJ( pLI"lI1d 1I11'rnhl'drlC' receptor on the mldgut epithella1 cells. ,lfflTlltv of thi' lrv:.lal IOXln:, veines from relativelj 10\'1 to high. An ,lltlVt' cl)'1't! of IPSCdl'Lh lnvolves the charactenzatlon of the receptor Wllldl, ln ',ornc' ld~)("., 1'-. belleved to bl' d glYlllsylatecJ prote1n (Know1es dlll! 111.11' , 198C, HofrnJIl11 ct al., 1988; Ellar, 1990). lt has been "uqq('",ted thdt rPll'ptor glvlosylatlon lS essentlal for tOtln blndlng and tll'Il ilv d~ldln:,t pdlhoty!'t' fi or Cry 1 g(.!ne product the tOXHl actIng like d lt'( t 111 (111.11'. 1990) 1Il l' .' (' Il d () t 0 X 1n C' VC' n t LI il 11 Y cau Se ~, the e p 1 the lia 1 ce 11 s ta d 1S ru p t bY f(llllllllll lvtll pOl'l':, ln the lnsect mcmbrane (LI et al., 1991). The tngger 1(1) tilt' PUI'L' fLlllllat lon l11ay hf' dup to receptar blndlng and the consequent llltl'i ,ll t 1011 of ttlt' tOXlrl Wlttl thl' membrane bllayer. The format 1011 of pores ln tilt' IIlPll1t 1I,IfH' (1f su'-.cl'ptlllle 1nsect ce11s lauses cl net Influx of Ions ,lnd ,Ill dlllllllp,ll1ylllll lnflow of water WhlCh force" the cells to swell and • 28 lyse (Hofte and Whiteley, 1989). Variability ln insecticida1 activity lS a150 ùttnblltl'd tn tlll' • susceptibility of the insect, v.Jlth three types being ldentlflC'd (~Illlll' d al., 1990): - Ty pel: l n sec t :: wh i c h S LI ff e r ~l e n e l' al pd r a 1y :, 1:' ,Ill li ~ htlI~ blood pH change to crystal proteln alone (Bonlbp mo,., ùlHl Nùnducd ~C\td). - Type II: Insects WhlCh are susceptlb1e to crystùb ,110nl'. exhibit gut para1ysls but no leakage, no chan~lP Hl blllOO pli, and no general para1ysis (most Lepldoptera). -Type III. Insects which reqllll'C both cry~tals dlHl ',port'" fOl pathogenesis (Anagasta kuehnlel1a and Lymantria dispar) . • • 29 REFERENCES • Angu~, T. A. 1956. Extrac.tlOn, punflcation and properties of Baei77us sotto toxin. Can J. Mlcroblo1. 2: 416-426. Anan., 1977. The carrot weevll. Agnculture Canada Insect Identification. Sheet no. 28. Aronson, A. 1., W. Beckman, P. Dunn. 1986. Baeillus thuringlel~sjs and relalcd lnsect pdthogens. Mlcroblo1. Rev. 50: 1-24. Balnes, U. 1 k ~tewarL and G. BOlvln. 1990. Consumptlon of carrot weevil (Coleoptprd: lurrul1onldae) by five speCles of carabids (Coleoptera: Carab1dac) abundant ln carrot fIelds ln southwestern Quebec. EnVIron. fnfomol 19: 1146-1149. BeaudOin, G. and G B01Vln. 1935. Effets d'accouplements répétés sur l' OV 1pO', 1 t Ion du c~drançon de l a carotte, LIst ronotu 5 oregonens" s (Colpoptpra: CUrcull0nHjae) au laboratolre. Rev. EntamaI. Québec. 30: 23 - 27 . Hechtel, D. B. Jnd L A. Bulla. 1976. Electron moctOSCOplC study of sporulaI Ion and parasporal crystal formation in Baenlus thunnglcns7s. J. Bactenal. 127: 1472-1481. • Beegle. C. C. and 1. Yamamoto 1992. InVItatIon paper (c. P. Alexander rund)' Hl(,toloqy of Bac717us thunnglens7s Berliner, research and devclopmpnt. Cdn [ntomol. 124' 587-616. Belair, Gand G ROlvln 1985 Susceptlblllty of the carrot weevil (Lalportpra (un ullonldae) ta Stelnernema fe7tlae, Steinernema blblonls dlHt flctcrorhabdTtis. heliothldlS. J. Nematol. 17: 363-366. B{lrllner, r 1911 UtH'r che Schalffsucht der Mehlrnottentaupe. Z. Gesamte Getreldcwes. 3' 63-70 Ikrnhard, K. 1986 Studles on the delta-endotoxin of BaeUlus thunnglens7s var tenebnonls. fEMS M7crub701. Lett. 33: 261-266. ROlv1n, G. 1985a tValuatlon of mon~torlng techniques for the carrot we(lvll, Llstranotos on~goMnsls (Coleoptera: Curculionidae). Cano Entama' 117: 927-933 BOlvln, G. 1985b Anaphcs sard7datus (GIrault) (Hymenopterëi.: Mymandae), an eg9 parasIte of the carrot weevll, Listronotus oregonensis (Le Conte). Cano EntamoJ. 118: 393-394. • 30 Boivin, G. and G. Bélair. 1989. Infectiv1ty of two strains of Ste1nerncma fe7tiae (Rhabd1tida: Ste1nernemat1dae) ln relat10n to tpmperature. • age and sex of carrot weev1l (Coleoptera: Curcullon1dae) ddlllt\. J. [con. [ntomo). 82: 762-765. Boivin, G. and P. Saunol 1984. Reseau de lutte integree: prO~lr,\mme dl' dépistage. Prad. Ayr/c. 7: 20-21. Boyce. A. M. 1929. A study of the b1010gy of the parsley stalk wl'pvil. J. Ecan. Entamai. 20: 814. Brodeur, L. 1985. Ellmlnons le charançon de la carotte. L'lIart1culture. 1: 15. Buchanan, L. L. 1932. The parsley and carrot weevil. Bull. Urooklyn [ntomo). Soc. 27: 7. Carroll, J., J. Ll and D. J. Ellar. 1989. Proteolytlc pro(e~sHlg of d coleopteran speclflc delta-endotox1n prodlllpd by Bacillus thurlng1ensls var. tenebrlon1s. B1ochem. 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OWl'WI!1tl'rlnq '>11('\ dlHI ~1It'V1Vdl nI the carrot wee"ll, Llstronotus or(lgoncn~l~ (Colpoptl'l'd CurcullOnldae). Env/l'on. fntomol. 15: 113-117. Grafius, E • M. Otto dnd 8 eolluls 1983 [)O\PltUH! .111(1 lontl'ol of ldlTO\ weevll. N/ch. St. Un/v. [xt. Hull. [-890. (PP. Hannay, C. L. 1953. lrystdll1np Hll111';lOn', III dflrotl1C '>pOl'pfOnlllll Harcourt. D. L. 1959. Insects of the ~l'ù~{)n 1958 ln ItH' OtL1W,! ,ln'c1. (dn. Ins. Pest Rev 37. 80-84, 156, 179. 196. Harcourt, D. G. 1963 Veqetahlp ITl',ects. Cano Ins. Pest f(ev. 41 913 • Harris, H. M. 1926 A rH'W carrot pP')t, WItt! noL(>', on II" 11ft' hl',lmy ,/ Ecan. Entamal 19 494-497 Helmpel, A. M. 1967 A crl1lcdl rpVIPvl of [)a(1ilu~ thunnglrn',f', Hf'rlln('r and othf'r cry~talld('r()lI:, bdctprld !1nn Nov Entolllul. 12 ?H71;>7 Helmpel, A t~. and T. A Anqw, 1958 lhf' tclxorHHTlY of HI'.prt p,tlho rel ated to Aac / Il us cereus f ran~ 1 and dnd 1 rrlflk 1,trld L ,JfI ,/ NlcrobJol. 4: 531-541 Heimpel, fi M and 1. A AnrJU::" 1960. On thr' LlX Of\ orny rd (('rtdlrt entornogenous crys ta 111 f ('rou '., bdC ter 1 il ,J. 1nsect Pa t ho 1 2 -~ Il "519 Henderson, L. S 1939 A reVl~.lon of Ulf' Cjf!rilJ', Il',tronatu',' (lurcullonldae' Coleoptera) Kan Unlv SCI. Hull 26' ?)r) 331 Herrnstadt, l., G. G. Sour!,:>, E P 1,>111(01 and [J s t r a ln 0 f Bac / Il U j t h U ri n 9 / en;. J r, vil t 11 cl (1 1 VIt Y d q d HI " t rr, 1(. () P t (. r d li lnsects. 8/o/Tcchnology 4 305-308 He r r n st ad t, c., 1 E. G1 10 r y, [) rI . ~ 0 b 1(!', kI, B [J Bp n fi P Il dT 1rj 1 Il • Gaertner 1987 tJuclr:otldr, '.,r!qupnu.."., and dr!dUlI:d arnlf10 dr ',Ij ',('(jU('TI((' of a c.oleoptr=ran-actlVe delta-endotoxin gene fram Baci77us thrulnglcnsls ssp. san dlago Gene (Amst). 57: 37-46. • Hofmann, C., P. 1 uthy, R. Hutter and V. Pllska. 1988. Bindlng of the delta-endotoxln frorTl Baci/lus thru/ng7ensls to brush-border membrane ver,l(lr~<; of the cabbage butterfly (Plens brassicae). Eur. J. Brochcm. 173. 85-91. floftr!, H. dnd H. fL Whlteley. 1989. Insectlcldal crystal protelns of Nacdlus thunng/cns/s. M/crobl01. Rev. 53: 242-255. lIolmr:'" K. C. and H. E. Monroe. 1965. Studles on the structure of pardsporal lnclu<,lOns fromBaci71us thurJngicnsls. J. Mol. Biol. 14: 572·581. !'.hlWdLl, ~. 1901. On a klnd of .;evere flacherie (Sotto disease). Da/nihon Sanshl Kalho 114' 1-5 l'.hlwatù, S. 1905 About "Salto Kin", a bacillus of a disease of the silk würm. nalnihon Sansh/ Ka/ho 160: 1-8. ]vlrl',klene, V 1978. Nonldentlty of lecithlnase and a-toxin from Baci17us thUrlfH]/cnsls [)('rl1ner 7eZ/sy Konf. Nolodykh Veh. Inst. Zool . Para7lthol I1kad I/t. US SR 2: 13-15. ,1.1hl1, N., W ~(hlldt('r and K Gplder. 1987. Cloning of an insecticidal • lOX11l gpnp of Bac177us thurJng/ensis ssp. tenebrion/s and lts pxprr c,l,lon 111 [scher/chla co17 cells. FEMS Nlcrobiol. Lett. 48: 311- 116. ,Llqllf't, r., H lIutter and P. Luthy. 1987. Specificity of 8aci77us thur1ny/ens/~ cJ(~lta-endotoxin. Appl. Environ. Microbiol. 53: 500- 504. ,)llIH"" Il fL, V. KdrLHldkdt-afl and H. D. Burges. 1983. Phages naturally <1~,'>Olldted wlth thp a/zawal variety of insect pathogen Bac/77us thulïI/91cn~/~ and theH' releva,'1cP to stranl idE:ntificatlon. J. Appl. nactl'l'Iol 54 373 377. 1\ Il (l\~ 1\' '> , B Il d nd D. J [1 1a r . l 986 . Cha r a ete r1 z a t ion a nli par t i al pUt'1 f lCdt Ion of a plasma membrane receptor for BacU7us thul'/nql('nSI~ Vilt'. kurstakl lepldopteran specifie b-endotoxin. J. Cel 1 5cI 83. 89-101 I\l'lt'l!, fi. 1971ù. Concernlng n-exotoxln produced by vegetatlve cells of fjac/llu~ thunngU!nS15 and Bac771us cereus. J. Invertebr. Pathol. • 34 17: 134-135. Krieg, A. 1971b. Is the potentlal pathogenielty of baeill1 for 1n5eet<; • related to productlon of a-exotoxln. J. Invertcbr. Pathol. 18: 425 426. Kneg, A. 1986. 8ac71/us thunnglensls, l'ln mlkrOD1('llps In~pktllld. Grundlagen und Anwendunq. Acta Phytomed la: 1-191. Krieg. A. and 0 Lysenko. 1979. Toxln::. dnd enzymes uf sevprdl SJJelle~ of Bac717us. especlall:{ of thl' [jacll/us ecreu!' thunng1l:,nsl.\ ~lrOlIp lb7 8akb. JI. Ilbstr. 134. 70 88 Kneg, A., A. M. Huger dnd W. Schnetter 1987d. Bael1/u~ thur1nqlt'I1!'I~ var. san dlego straln M-7 lS Hlentlca1 to the formtTly 111 l;pmdny lso1ated straln BI 2S682 EL t. <,sp tcncbnol1/'> wllllh 1\ pathogenlc to co1eoptpran lfl<;ects. ,} Ilpp/. Entomal 104 4114?4 Kneg, A., A. M HuçjPr. (; A Ldnqenbrllch and W ~(hn('ltl'Y' 19R~ lidcIllu\ thun ng7 ens 1 s var tcnrbn on 1 S' f~ 1 n npllpr, ljPql'nllhpt' I.\t'vl'Il von Coleopteren wlrksamer Pathotyp. 1 angcw lnt 96 500 ~OB Krieg, A., W. S,chnettcr, A M HUSF'f' dnd G. A Idnqpnbru(h 19871> . Baeillus thunnglensls ssp tenebnonls ,>traHI BI 256 R? thlnl pathotype wlthln the H-serotypr BA88. Syst App/. Mlerohlol 9: 138 • 141. Krywienczyk, J. and T. A. Angus. 1960. A sero1og1cal COmpdr1',On of thp parasporal bodIes of three Inseet pathogens. J. Inseet Pathol. 2: 411-417. Krywienczyk, J., H. T. Dulmage and P. G. fast. 1978. (]ccurrpncp of two seroïoglcal dIstInct groups wlthln Bacll/u!' thur1nglcn"/,, '.f'rotypp 3 a, b var. k urs t a k1. Il. 1n ver t c br. Pa t h 0 1 31. 177 3 7 5 Kurstak, E. 1962 Donnpcs Sllr 1'PPlloOtlf> hdCtf'rlPnnp Yldtllyp11p prOVOqllPP par un 8aelilus du type [Jc1clllu~ thurlng/cf/5./:' '.ur Iphy',t/d kuhnie71a Zeller. Entomophaga Mem. lion ~er. 2' 245-?47 Lambert, B. and M. Pefpfoen 1992 In~(!ctl( Ida1 proml',p of lJacillu') thuringlcns7s Blose/cnec 42 112 122 Levinson, B L.• K L. Ka<.,yan, ') S ChIU. r. C (urner and ,J M. Gonzales, Jr 1990. Idf~nt1fllatl()n of r~-PxotOXHI productIon, plasmlds encodlng I)-exotoxln, and a new b 1Il t h y, P., .J Cordlpr dnd fi M. FIscher 1982. Baci17us thurlng7ens7s a', cl bdctf'rJdl In,pctlclde. bd51C consIderations and appllcation. In- Mlc.robldl and VIral Pestlcldes, ed. E Kurstak. pp. 35-74. New York: MrlrlP 1 [)r~ kpr. 1ne. Martel, P., L. 1<. HaY'ns and fi J. Svec. 1976. The llfe hlstory of the Cdrrot wPPvll, / Istronotus oregonensis (Coleoptera: Curculionidae). Can Entomol. 107 471-475. Mdrtpl, P., H J Sv!'( Mllnl', R , A. ! (;1', D. Rlvers and D. H. Dean. 1990. Speciflcity of lnsectl( IU,ll cryc;tdl protelns. In: Analytical Chemlstry of Bad77us thunnglrnsls, pus. L. ,~. Hlckle and W. L. Fltch. pp. 22-35. Wdc;hlnqton D. C.: Amerlcan Chemlcal Soclety. Mohd-Saleh, M. B., C. C. Beegle and L. C. LeWIS. 1980. Fermentation media dnd productlon of exotoxln by three varletle~ of Baci17us thunnglcns15 Ll. Invcrtebr. Pathol. 35' 75-83. N1Lkprson, K W. 1980. Structure and function of the Bacillus thunng7ensis proteln crystal I3lotechno7. B7oeng< 12: 1305-1335. NOtT1S. J. R. 1964. Ttlt' classifIcatIon of Baci17us thunngiens7s. J. App7. Bacter/al. 27' 4j9-447. • 36 Pepper, B. B. 1942. The carrot weevil, Listronotus latiusculus (80tH'.). 111 New Jersey and its control. N. J. Agric. Exp. Stat. Bul1. 693 1-20. • Pepper, B. B. and L. E. Hagmar,l'l. 1939 The can'ot wc('vil. L Ist,'onotu~ 7atiuscu7us (Boh.), a new pest on celery. lJ. [con. Entamai 31 ?6? 266. Perron, J. P. 1971. Insect pests of canots 111 or~ldnll \o1L of southwestern Quebec wlth specIal reference to ttll' Cdrrot wl'pvll, Listronotus oregonensls (Coleoptera' CurclllIOnldal'). Cali. Intomol. 103: 1441-1448. Poinar, G. O., Jr. 1989. Fxamlnatloll of npoaplertdtl1 POlnar, G. O., ,li'. 1990. laxonurny ùnd blOlogy of Ste HlPrml't 1(1<11' ùrHI HeterorhabdltlClùe. ln fntomopathoqclllC NemJtod(~\ Hl BlOloql Control, eds. R Gauqlpr and H. K. Kayd. pp. 23 61. B()~lt\Jll: (I{( Press. Rlbler, J. and M. M. Le Cadet. 1973. ttude ultrastruturùlp pt (11l1't Iqul' dl' la sporulatlOn de Bacl1llJs thUrlnglcnsls var. b('rllnC'/' 1715. Remarques sur la formlllùt lon do l'lnclu'.lon pard\pOr,llf' /Inn. • Microblol. 17' 373-375. Rietmuller, U. anrl G. A Lanqpnbruch 1989. Two blO the efflcacy of BaC/llus thur/nglcnsl~ ~,sp. tencbnorl1s clCjclln',l thp larvae of the Colorado potatoe beetle (lept,notarsa dccem/lnrata) Entomophaga 34' 237-246 Searle, 1. and W. N Yul(~ 1988. fUllgal control of thp (,Irrol wppvi 1. Proc. XVIII lnt. Congrcss Entomol. Vancouver, B. C p. 26? Sebesta, K and K. Horska. 1970. Melhùnlsm c, of lnhJlntlon of DNJ\ (jppl'ndpnt RNA polvmera,;f' by f!Xotoxln of Hacll/us thunnguJw'/) /f/ochrm. 8lophys. Acta 209' 357-367. Sekar, V. 1988 The Insectll1dùl crystal protel11 qem: l', exprp',',pd tri vegetatIve cells of Bac/llus thur/nglcnsls var. tcncbr,onl~. Curr. Microblo1. 17: 347-350. Simonet, D. E. ùnd B. L Davenport 1981. Temperature n~qujreml'ntf, for development and OVlposltlOn of the carrot weevll. Ann. Entomol. Soc . Am . 74: 312 - 3 1 5 • 37 Smith, R. A. 1987. Use of crystal serology ta differentiate among varieties of 8aci77us thuring/ensis. J. Invertebr. Patha7. 50: 1-8. • St. Jul ian, G., L. A. Bulla and C. W. Hesseltlne. 1971. Germinatlon and outgrowth of Bac/17us thunng/ens/s and BaC/l1us a7vei spores viewed by scannlng electron and phase-contrast mlcroscopy. Cano J. M/crob/a7 17 373-375 Stevenson, A H. 1976. Sea~onal hlstory of the carrot weevil. Listronotus oregoncnsis (loleopterd Curcullonldae) ln the Holland Marsh, OntarIO. Proc lntamal Soc. Ont. 107: 71-78. Stpven')on, A. fL 1981. Ldrrot 1f1C,ects. Ontano Mlnlstry of Agnculture and lood 1 ae t Sh(>f't No 81 -007. Stpven~on, A B. 1985 ! ady warnlng system for the carrot weevil (laleoptt'rd LllrLllllon1ddf~) and lts evaluation ln commercial carrots ln OntarIo. J'con intamal. 78. 704-708. 1oumanoff, C. 1954 L'iH tlOn de Bac711us cereus var. alest1 Tourn. et Vago sur 1re., chcnlllf:s de Galler/a melonella L. and Hyponomeuta eognatclla. ~nn Inst. Pasteur Paris. 86: 570-597. Whllcomb, W IL 1965 lhe carrot weevil ln Ma~sachussetts: biology and control Mas" /Igne. Exp. Stat. Bu7l 550: 1-30. • WlClller, W Rand H R Wtlllpley 1989 fwo h1ghly related insecticidal cry<;!a] pr'ot(,ln~> of Rac7llu'l thunnglensis ssp. kurstaki possess dlffprent host l'dn~F~ Sr)(!Clf,cltH~s J. Bactenol. 171: 965-974. Wu, D. and F. N Chiln~l. 1985. Synerglsm ln mosqultocidal aetivity of 26 and 65 kDa protrln:) tram Rac71lus thunngiensis ssp. Israe7ensis cry<;tal FERS lctt 190' ?32-236 Yamamoto, J. dnd R Mclaughlln. 1981. IsolatlOn of a protein from the paraspordl lry\ta] ot Bac1 llus thurlng/ens/s var. kurstakl toxie to the mo<;qulto larva, Acdcs taen/orhynchus. B/ochem. B/ophys. Res. Commun. 103' 414-421 lehndpr, G W. and W 0 Gelernter. 1989. Activity of the M-ONE formlJlatlon of a rww stra1n of BacJ7lus thunnglens/s agalnst the Colorado patato beetle (Coleoptera: Chrysomelidae): relationship belween suscept1blllty and lnseet life stage. J. Econ. Entomol. 82: 750-761 • 38 • CHAPT ER 2 Response of the carrot weevil. Listronotus otcgoncnsis (Coleoptera:Curculionidae). to stralns of Bacil1us thurlnglcn~,s. • • • ABSTRACT Mortallty and frass bloassays were done ta investigate the toxlcity ()f ','!ven slrdln", of 8acIllus thuringiensis against the adult carrot V/f! (.! V 1 l'" L/5tronotus orcgoncns7s. A seml-artlflclal diet of carrot follage wlth 4% agar W1S ~elect~d lü maXlmlze feedlng by the insects . 8acillus thunng/(~nsls subsp. tcncbnonls (8TT), and two unldentlfled B. thur7ng/cnS1S stra1ns, A30 dnd A429, gave the lowcst LOs: valurs. The frass hloas',tly supported the concluslOns of the mortallty assay. MOt'tallty of ,1c1Ult', continUE!d after thelr removal from the lnsectlcldal medlum. with ttlf> hlqhf!':.t llIortallty t)(>HI~ cdused by stralns A429 and 8TT. Survlvors From thf> frd','; b10rl',·'dY. Itl1tlillly expüsed to stralnS A30. A429. and BTT, did no! tl'~unll' lIoY'mal lpvl'l~ of feedlng after their removal From the IfI~(,('lCldal fTH!dllJlll Attempts to relsolate the B. thunngiensls 1I11( t'()Orqanl~,ms frolll thr expenmental lnsects revealed that the carrot v/f>f> V 11 hdrbolJn B. thunnglensls-1ike arganlsms in its gut and on ather • " t t tH t IJr(", • 40 INTRODUCTION • The carrot weevil, LJstronotus oregonensis (Le Conte). 15 a major' pest of Umbelllferous plants ln the northeastern 5rctors of Ull' UnIted States of Amenca (Chandler. 1926: Peppcr. 1942; Wtlltlomb. 1965, (;t'dflll~ and Otto. 1979) and Canada (Pen'on. 1971; Mdl'tel et a7., 19/5; Stt'vpmon. 19 76) . It a tt a c k 5 cel e:''y (P e pp e r a nri Ha ~l ma r, n. 1938) and pan 1(' v (H 0 Y\ (' , 1927). but lts prefeneJ host lS thr carrot wlth wl'l'd '>P('( ll'\ \llch ù\ wild carrots (Daucus carota L ), plantuln (Pldntaga ~p), dlHl dol!<, (UunJC'\ s P .) al sos e r v 1 Tl 9 J S ho s t <, (P Ppp (> r , 194 ? Hy c; (' t' , 197 5 ) The adult femalp IdYS e~lq<; ln ttlt' ~lPt loll' and (t'OIoJn of thl' canot. The larvae, upon Cl1lpr'lpncp, tunnel lnto thl' toot wtll'rp ttH'Y fppd Jnd develop untll thf'y eXIt Into thf' sOlI Jnd pupatp (Prrron, 1971) Tlw carrot weevlls overwlntpr ln thr sOlI ih adults Slnce 1967, (ùrrot root H1Jury Cdll"l'd by the (ùrrot wPPvII rarlCjl'd from 2 - 22~~ 0 f thr lotù 1 crop prouuct Ion 1 n thp QUf'lJe( r(l(j Ion" of Shernngton, St -Mlch(~I, ilnd <:îto ClotIlde (Perron, 1971) wlth damdqr of up to 40% of the carrot crop whon Ipft u[)r()ntrollpd (BoivHl, 1985cl). Thf' packlng and cannIng Indll"trl0" tolpl'atl\ no morf' thdll 5% tool H1Jury • (Perron, 1971) whlch makps df J( IPnt (oJitroi nj(~d~lIl(~'. rH'CP\',dry The only control methoo avallrto10 for thp carrol wPPvll l', thl] use of follar Insectlclde c, aCj (BOlVlIl, 19[,5a). H(),.H~vpr, th!! dpvplnpmr>nt of rf",I',tdrl(P 10 (hpmlCal lnsectlCldes by many pP')t IWJr;ct~, dnd ttw CjPtl{!rdl (on(pYrI fI)) PrlVlr'onmPllt damage have resultprj ln thf' d!!v!~lopml!nt of dn lntl'qrdtpd pP',! I1Idfld!jP!TlPfll program wlth ornpl;d~,I:. on alterndtlve control mrthod'. lhr>rp l', Ilttlf' Informatlon on alt(!rncltlvP control stralegll", HI thp llterdtlln' 10 (j,JI!', an egg parasltoHJ, /lnaphcs sordldatur, ([lOIVlfI, IfJF35b) Mid pntO[fl(HjPIIOII', nematodes, Ste/nerncma carpocapsac [,5 frltlJ(! (Polnar, ]gB9) l 5 felt/ae [=5. blblonls (Polndr, 1989)), Mid lIetcrorhabdltl~ bacterTophora [=11. he710thldls (Polrlar, 1990)J (B(~J;l1r Mid BO]Vlrl, IfJB5, BOI>/HI ,Uld Bel a lr, 198 9) h a ve be e n f; xa m HI ~ d The entOln0pa t h(Jgr~n 1 C bar: tCrl um, Bac 1 Il us thurI ng 1 en') i s, O( r:uP] P', an lmportant posltlon ln thp fJeld of bloloCjlcal control [J. thunnglcn515 is a Gram-posltlv~, enrJo::;pore-formlng, :..oll-dwelllng bactf!rlUm WhlC.h • 41 produces an insecti' 1dal cryst~lline proteln (ICP, also known as 6- endotoxln) dunng spcrulatlOn (Hafte and Whiteley, 1989). Once ingested • by susceptible 1nse ts, the ICP dlssolves in the midgut and is proteolyt lcally actlvated to a smaller molecular welght toxin (Lilly et al., 1980) ThAse tox i ns bi nd ta receptors on the brush border membrane of the ITlldgut pplthellal c.ells (Hofmann et al., 1988a and b; Knowles and [lldt" 1986; Hf:ndrickx et aÎ., 1989), causing cell lYS1S, feeding lnhlbltHHI, dnd eventually death (Fast, 1981; Krleg et al., 1983). Pr2vloU'> 1 y (]pscnbpd straH1S have demonstrated aetlv1ty against h:.!pldoptpran and dlpteran C;prCI!?S (Kurstak, 1962; Goldberg and Margalit, 1977), More r2u~ntly, [3. thunnglensls stralns taxIe to economlcally IITlportdnt pe<;t~) of thp ordpr Coleoplera have been dlscovered (Krieg et al., 1983; Herrmtadt ct al., 1986). B. thur7ng/cn<;/) <;lJhsp tcncbrionis [synonymous to B. thuringiensis sllbsp san d/cgo (Kneg ct al.,1987)], a strain pathogenic to Coleoptera, toqethl!r wlth :, IX lJrlldpnt lfled B. thuringlens7s strains were used in the (urrent ~tudy. 1hc undf'flTlrd stralns, lsolated from Canadlan soils, were shawn ta b(~ actlVe dg MATERIAlS AND METHODS Chcmicals Manabas 1 C and dl bas i c potass i um phosphate (KH 2P0 4 and K2 HPOd, dlthlOthr"Cltol. phenylmcthylsulfonylfluoride, Ampicillin, Polymyxin B, and SOdlllfl1 carOolldtl' (Na .. CO,l werc purchased from Slgma Chemical Company (MI~~()UI'i ,USA). Nutr1rnt ùgar was purchased from BBL (Maryland,USA) and the Bio-Rad proteln assay klt was from BIo-Rad (Ontario,Canada) . • 42 Hacteria1 cultures and spore-crystal suspensions • Baci77us thunngiens/s subsp. tenebrion/s, and the SlX unldpntifH'd stralns of B. thuringiens/5 coded A30, A299. A311, M09. A410. and A429, were supplled by lmpenal Chrnllcal Industries 81010g1ca1 Prodllct~. Not'th American Technical Centre (Ontal'1o. Canada) li1€' Bac/llus thul'1nq/C'f1s/<; strains were grown on nlltnenl agar t'dffrred (pH 70) wlth t'qllll1lolar concentratlon<; (O.05M) of Kil/PO. and K"HPO,. After 5 days 111l11b,1t IOIl dt 30 oC, the bactenal lawn, ba:;rd upon mlcroscofllC l'XdnllnJt Ion. COfl'-.l\lpd of vegetatlve cells, sporan~11a, ~pore<, and rrY'.tah. Thpsp \t.HjP\ WPt'p harv2sted by scrap1ng, lyophllllCd, dnd <..ton'd at -20 "C. Spore-crystal sllSpOnSl(ln'. of l'ach "tra1n wpn' u<,pd ln toxicityassays To en~lln' that tOX1Clty nH',lSUY'OIlll'nt" wOllld not rdl!'(! crystals contalnlll9 dlfferpnl ,ul1ounL of proteine;, tfH' (Ollcpntt-.1I10n of crystal proteln for Pélch tJaclrrlal "tT,lln W mixtures were sllspendr:d 111 0 Ol'/a lrlton X 100 (v/v) (,(JIu! lOri ,Ifld wd',tH'd three times by cen1nfu9dtlOli (11,750 xc), ?? "r, 3 min) Ih(l flnd1 ppll<,1 was resuspendf'd ln il crystal s()lulJlll/1n~1 huffpr INd/O" 4 l q/l; phenylmethyle;ulfonylfluoY'ldp (<1 prote,l',f1 lflhlbltot 10 pt'llvPlIf ICP • degradatl0n), 87 mg/L; dllhlolhrpltol (to n'dlj((' dl',ulfld!' bond")' lS.4 rr.3/L; pH 10.0] ilnd so1ublI1l('d at 42 or by lnrubdluH! for? h, vorlpxllHI every 30 mln The sur,pl'n',lon Wil', untnfuqed (11,750 xq, 2? °(,1 nlln) and the supernatant assayed fur total prot(~Hl dCcordHI(1 to tld' BYd(lfoj'(1 I1lplhod (1976) uSlng d BIo [(dd proteln d~Jsay klt_ (BIO f protein assay 15 bdsed on d calour ChdrHJI! of Uw dyr!. th!' op! I( of the resultlng solutIon lncreaSH1~j Wlth ln(red("n~l (on protein. Bovlne S(~nWl dlbumui wa c usrd as il 5landdrd. Co lony and reari ng comll ti OTIS Carrot V/eRVl!:. dvalldble ln laboratory lulturp V/l'n' IH!rlodlCrllly supplemented wlth weevlh from Aync.ultdn' Canada f<1~'J(:dY'Ch 'JtdtlOri (~t. Jean-sur-Plchellf~u) to rnalnlaln hybnd v Iyor Thf> colorlY Wd', y('dYf'd ln incubator with a photoperlOd of 16 h (Mdrtl:1 ct al, 1975) MId d temperature reglme of 24:16 oC (Day:Nlqht). Ihp ddult'J v/(~rr.: pltlled ln • 43 .. Jar~ contdlnlng carrots, the carrots serving as bath food and oVlpositlon .-,itt"... After the lnsects oVlposited in the carrots, the carrots wet~e • lransferred to a plastlc box having a screen on the bottom and this box was then placed inside another whlch had a SOlld bottom Once the larvae (!mprgpd they dropped to the lower box from whence they were co 11 ected and pldled Hl mOlst sterile 5011 for pupatlon and adult emergence. These rldu l t" w(!re used ta bath ma 1 nta 1 n the col ony and for experi menta 1 pllrposes. Selection of diet maximizing ;nsect feeding lo seled il dlet that would be avallable year round, and act as a qll',tdtory ',tlmulant enSUrlnq uptake of the lep placed on lt, six diets WPyp fed to the .ldtllt insec.ts:(l) carrot root, (2) softeneu (steamed) LtlYTot roof wllh 2'1:, (w/v) .lgdY, (3) fresh follage, (4) follage extract Lonld 1111n9 4j~, (w/v) .lgar, (5) fol iage homogenate ln 4% (w/v) agar, and (6) 4''%, (w,lv) aqtlY' DIPl'. 4 and 5 were prepared by suspendlng 22 9 of chopped folldql' in 70 ml of water rOllJ~le horno~jenates consisted of the fibrous dnu ~olubl(> port Ions of the sllsprnslon whereas the extract was rendered • low Ifl fl!JPt tly ~Irdvlty s~dlfnentatlOTl. Equal volumes of autoclaved agar Wl'tP I\hllnddnl food WJ', plaLed Hl pld~tl( Petn dlshes (15xlOOmrn dlameter) wlth 111()I',1 fllter pdJlPI lo wlllLh JO adulL wcrL' added The plates were placed ln till' Inluh,ltor::. dncl thl' nurnhpr of fra,;::. pellets counted dally for three d Inscctlcidal Bloassays l\'IO dl fferent bloassays were done ta measure the toxiClty of the "Il cl Ill', iHldltlst Hw edlTot wepvll' one based upon adult mortallty and the (llil!'l d I11lHllfHùtlon of the ft'dSS productIon assay of van Frank.enhuyzen ,111lf l;l'lnqllrtpll (1991) regardecf as a rapld method to determlne the '>Pl'C1flllty of JlUllll'l'OU~ B. thunnglensls toxlns against Lepldoptera. [)lPt 5, the OptllllUnl medIum for lnsect feedlng, was offered to the • 44 lnsects as food cyllnders (6x6mm d1ametcr; 10 cyllndrl's/plùte) lut frolll foliage homogenate supplemented w1th agar (Chapter 2. "Select ion nt uÎl't • maxim1z1ng lnsect feedlng"). The cyllnJer:, wcrc coatrd wlth tl,~t ~pOl'l' crystal suspenslons by dlpplng thom lnto the Jlffel"l'l1t do<.,('~, (lf l'l'hvdr,lIl'd spore-crystal mlxtures ln dlstllled water. dgltùtlnq tlll' PI'l'P,U\\tlllll\ tOI' 5 sec and allowlnq them t(, alt'-ury. The ICP do\e~ lr1l11ldl'd l'J. l'JO. N5. and 300 Ilg pl'otel!ljrnl 01stllled watE:r ~erved ,1) J control Adults, collected 3-8 ddYS after ()clo~HHl, wprp addpd tn tlll' <11P\ ln Petrl dlshes (15xlOOITli1l dlameter) CO:lLllr11nu 11101,>\pl1l'd f 11\1'1 pdpl'\. Insects were allowed to fcru on Dlet 5 for 8 (LlY'> III \Ill' lllo\·tall\Y bloassay, and for "lX days ln the frass bloas~dY both ,It ?5°l III ddt'knl'~\. Adults were treated ln ~lrOllps of 15 ln')ect~ (8 IS. 7 dS) ppr plat!' wllh .1 total of 60 adu 1ts pel' dost' Mortal1ty was monltorrd dJlly over the dSsay ppr\od ,lnd th!, I11P!lldll lethal dose (LD ,,) fOt' rach tp5,t .,traHl was cal(ulJ.tpd hy Ill'ohit .trldlY',I~, (Finney, 1971). fhe cumuldtlVE.' r!pcrcùsp ln nllmhpl' of ft"t'.', p!'11!·t" p!'t insect exposed to the Samf} lep J()<,(I" ovpr cl !lPY'1o!l of (l ddY'. Wd', calculated. ThlS dpproach devI,!tpd From that of Vdll frdnkflllhIlY/{'lI ,llld Gringorten (1991) Hl Wh1Ch the numbpr of Hl\ect., Lllllflll to pIO(\Ut!· 11'.1', .. • pellets was recorded Thp COllCf'ntratlon (lf pl'()tf~lll r!'(juII'f'd 10 1111111>11 frass Py'OdUc.tlon by 50~o (FP"J wa~. determuwd Inseet survival and frass product ion aftcr rcmoval From thl' d/l't contalning lep Lepldopteran larvde are known to survive Intoxlcatloll hy /1. thuring7ensis (Mllne et a7., 1990). The' c.onSf!(jupncp of llrrtltf'd lntoxlcation for beetles 15 not known Bpcau';f! UIP ',lJrvlvdl of 111',1>1\', formerly exposed to B thurlngu:nsls lnflllpn(p', oVf'r,t11 f!fflrdf,y of UI!' lnSectlclde, lt l'IdS necessary lo det(lrrlllnf~ thi> lonq tPY'lrI (lffprt of U thunngu:ns7s stralW, on arJlllts pr p '/lolJ',ly r:I[JO':,(·rj to UI(I [f,P dt thf' [f)" values Survlvors wlthln d C.JIV(~n tn~(jtrnf;nt, dt B ddY'. for mOY!.III!; Mid f) days for fras::. productlOn, werr~ COWj011ddV~d on unlredtf!d food (/llnr!PY', ln Petri plates ~llth mOI"ten0d flller pappr. rhr: Irl',(:rl', vJ«r l' IrtCllbdtl:d at a temperature regHne of 25.16 oc (Day:t~H1hl) wlth d 16:e h (Udf IllfJhl.) • 45 photoperiod. Mortal ity was recorded over a period of seven days and the • frass was counted for flve days. Detection of B. thuringiensis-like bacteria Control ddult~ e/hJtJlted mortality. Therefore, to confirm thp vdlldlty of mortallty of Insects trealed with the B. thuringiensis strdln~, Koch'" postulate wa'} Inltiated. B. thunnglensis or B. thunngu'nsls Ilkp orydnl~,rn:) were detected ln treated as well as control HI',Pct:) )(rt!f:nlrlg for possIble contamlnatlOn by B. thunnglensls of the tontrol In\ect~ WdS done Several untreated Insect body pa~ts were smeared on two nwdla NAP (Nutnent dCJdr -t 5/Ig/ml Polymyxln B sulfate + 4f.Lg/ml I\mplc Illln) whlCh L1VOY") (OlTlpptltlve growth of B. thrunnglensls relative 10 othPr IIIlrY'(J(H'qJfll',rn", dfld NI\ (NlJtnenl agar) 2S d control. Both media WPt'l' ',IlIJ')eqllpnlly IrlCllhJtpd dt 37 oc For 4 days. NAP was nlodlfled from NPP (Nul r If'nt d~ldr t ~/L~lIf1Il Po lymyxlll B sul fdte + 4/lg/ml PeIlle 1111n G) (Saleh ct al., 1969). lhp <,tructIJrec, r1at(ld on the medIa lncluded larval gut; gut trom unfpd adults, canot frd adults, and carrot fcd adults from another locallon ()t Jf:dn-~.lIr-I{lçhelH'u), hilpmolyrnph, and elytrd. fo check for • ttlP I)(l,><,i!llp ~Oln'l(l of contdnllnatl0rl, Cdrrot~ dlld ~oll wcre washed with ~l(>rll(l dl:,!lllpd w,ltpr from whJ(:h an allquot (40 i~l) was spread on the IlIPdid pldlps B. thurJnglcnsls-1lke (Jr~Janlsms were ldentlfled by the Snl1rnoff dlfferentlal )talll la detect the vegetative cells. spores and crystah (~mlrrlOff, 196?). Data analysis Thp LD.", values (medlan lethal dose) were calculated uSlng probit an,!IY:'ls (Flnnpy, 1971) The FP"o values (dose inducing 50% decrease in trI)'):' productIon) w('n~ calculated by llnear regression analysis of the fras:, blOi1',:,ay oald Statlstlcal analysls of palred data was performed USHlg Studrnt's t-tE'sl One-way analysls of variance (ANOVA) in conjunctlon wlth lukey's te,>t was used ta com;:>are groups of means. 5tat1st1cdl ~lgn1flCJnc(' was taken at p RESULTS • Diet selection To select for the diet conducive to food consumption. thl' l'lfl'ct 01 food formul ae on adul t defecat i on rate was dE't~rllli nrd Carrot <; d 1\1 Ilot ellcit frass pl'oductlOn to the same extent dS fn'<;h fol1iH]p (Ill! 1). However, ln both cases the add1t10f1 of tl~l HOlllogenlzlng the fo11d<]e and addin9 d~Fn' <,trol1qly pnh.1IHt'd 11',1,,<, productlon with d1et 5 (folla<]e homog0lliÜ(' wlth 4':., dqar) bPIIllj opl 1Il Il 1111 loI' frass prodllctlOn whrreas agar .::10f1(1 wa~) UH' 11'<1<.,1 pf fpl t 1Vl' I\lthllllqh Ilot quantifled, it was observed that the 1llS(lcts attJckl'd dll't 5 ",or!' ,lvldly than the other formulatlons. Determination of crystal protein content To standardize the alllount of insecticldal protp1n<; of UlP dlffl'n'Ilt strains for use lT1 the bioassays, the protein rontrnt of ttlf' u'y·,t.d', Wd', deterlllined (F1g. 2), Th(! rrsults varied wlth thr l,;olat(l', t',lIlCl"HI fl'Olll 2.11 ± 0.063 1/9 proteln/ mg sporr-crystal ll11xture for Il. lhurln(l1Cn''/', • subsp t enebn' on 1 S to 100 6 1. Il.07 Il(j prot/' 1n/ mtj ~porf' (ry', 1li 1 III 1 Xt III f> for straln A410. lep cffects on insect mortal;ty and frass production The effect of B. thunngiensls strolns on ddult wel!vll IIIOYtdilly estab1ished the lowest LD~[Jvalue for straln 1\429 onel UII! hl(jhl",t vrllup for strain A311 (Table 1). The rank arder of tOX1Clty for thl~ diffPYl'nt stra1ns was 1\429 ~ BTT ~ A30 / 1\299 A4l0 / A409 - 1\311 1 IqlJYI' l shows dose-respome curves of Ulf' rno',t tO/.ll <,trdlrt', dlld ri Il''.', tO/lf lsolate for comparlsoll purposeJ. Thf! rnost tOXIC strdlll', dl',pldY"d re1at1vely steep slopes vil th strain BTT showlrlg th(! sl.eer)l!',l Jlopp v,lIlJp (1.80 ± 0.25). To further asses::, the tOX1Clty of the B. thurlnglcnsl'J ',t.rdlw" • 47 • fIgure 1. The effect of food composition on adult rate of defecation. l, carrot root; 2, carrot root with 2% (w/v) agar; 3, fresh foliage; 4, foliage extracts contalning 4% (w/v) agar; 5, follage homogenate ln 4% (w/v) agar; 6,4% (w/v) agar. (N=3) . • • 48 • >0- c "C 160 fi) ~ "u 140 CD fi) c 0- 120 0 ...- 100 ln "~ CD 80 • CD a. c 60 u CD '1- 40 '1- 0 L.. 20 CD .Jl E ::l 0 Z 1 2 3 4 5 6 Food composition • • 1 1 • • 50 • • 1-- 1--m en N ...... 0 .- ...... 0 ~ 0 0 0 0 0 a a N 0 co CD 'lit N '"""" ~ • • • • Table 1 The: r:-edlan lethal rjose (LDs: ",g proteln Iml) for adults of the carrot weevll. L7stronotus oregonensls, exposed to deslgnated BacI77us thur7ng7ens7s stralns for 8 days at 25°( --~------~-----~------~~~~-=--~------~-~-==~-==--~--~~------c Straln LD~:,a 95"~flduclal llmlts Slope (,.g proteln:ml) LLg proteln/ml) ------~-_& A429 102 52 - 200 0.71 ± 0.07 BTT 118 89 - 155 1.80 ± 0.25 A30 143 101 - 204 1. 38 ± 0.11 A299 230 143 - 370 1.01 ± 0.05 A4IO 255 196 - 331 1. 90 ± 0.08 A409 4L9 201 - 914 0.64 ± 0.02 A311 461 266 - 800 0.90 ± 0.03 _E ____ =~~~ ___ = __ g~_=aa=_~ __ a=Kg~~~==3_=_~~ __ ~ ______~---~---~=~------a N=3 b Mean± standard error of the mean • Flgure 3.(A) Dose-responsecurves of themost vIrulent -;\1'<1111',,1\30, J\4?9, and BTT, compared to strain A311 which has low vHl!lprHP, (B) Correspond1ng probit regression linps. The data l'cprp'>l'nt,, 111l'.1I1'. ()f 3 morta lit y bi oa s says. P repre sents percent k 111 • • 53 A 100 • A30 • " A311 ~ A429 6 o BTT 80 ~! ....>- 60 c ...... 0 ~ 40 i ___ y-f " -y ~ /' 20 " 0 50 100 150 200 250 300 350 • Dose (11-9 protein/ml) • A30 8 A311 0 6.0 ".. A429 0 BTT r=O.91 0- '+- 0 5.5 r=O.97 +- r=O.98 .a 0.... a.. 5.0 r=O.92 4.5 ~------~~~~----~----~ 1 .75 2.00 2.25 2.50 2.75 • Log dose (J.L9 proteinjml) thelr effect on feedlng lnhibltlOn. as indlcatNi by a dt'lTt'cl~1' 111 t1'<1\\ productlon, was determlned. Stralns A429 (F~\\,= 10.67 i 1. 70 I,q pl'ntpln/ml) • and 8TT (FP:.=41.94 ± 4 20119 protcin/rnl) CJusl'd tho gre,ltt'<;t dI'C),fl,I\P 111 frass pellet productlon followed by strall1 1\30 (fr.",=- 94.58 t g.80 11<) protelnjml) (Flg.4). Straln A311 (f-f\,,= 1234.37 t 185.15 l'~l pl'nlpln/ml). however. was not S19tllflcantly dlfferent fro::1 ttll' contl'lIl ~lrllllp\ Ihl'l'!' was a slgnlficant correlatIon betwc>en Insl'ct lllortal dv ,1I1d l'l,dUt 1 1011 III f ras SpI' 0 duc t ion (r =- 0 99) (F 1 g. 5). 1\ t al 1 d 0 ,~ 8TT, al Id A429 showed li Sl~ll1lflcant dOc.Tea,;!' 111 Ih(' 1,,1lt' {lI 11'.1'>', pl'I Il' 1 production ppr lnscct over J P(ll'IOd of 6 days as l'Olllpdl'l'd ln ItH' 11'\-, t 0 X lC S t ra 1 n 1\ 3 1 1 F1 9 li r e 6 s h 0\'1 S a r (1 p r (' " (' nt a t 1 V (' r C ,; III 1 {l f Ill!' 1 11 f 1lIl' 1H l' of selecteJ stl'ains of B. thuringicns7s at 011P dO')ll~lP (2?5 IIC] pI'01(,111/m1) on frass product Ion 1h0 nlllllhpl' of frd'.', ppll!'I', p!'l' 111',('( 1 (lXIl()',I'd 1 () strains A30. 8TT, and 1\429 exhdntl'd cl 3-4 f(Jld IntoxIcation. Convel'sely, the dl'cr1'a~,e HI nunÜll't of pI'IIl'l'l prodlll('d pl'I ltlsect exposed to strilln 1\311 WJS not siqn1f1 lnseet 5urvival and frass production after rcmoval From diet conLdlnlnq • lep When adult vleevlh survlvlny ar ID.,' dOSdyC fOl cl IjlV('!1 1(1' ',Ildlll viere placed onto untrpcltl'd ulet 5 for 7 ddy'" IIlorldllly lorlllflUP!l lOI stralns A?99, AllI, A409, A30 and A410, rrrurLlllly vld', Ilol ',I surVlvors prevlously ':.ubJec..teu lo strdln:, !\4?9 dnd BI! t'dll!j(·rj Il[>lvll'l 5 5~~ Survlvlng ddul t:, prr:vlou~)l'y ')lJtJJP( lf!rj to 1 [J,,', rJWIl.1 .,tralns A30. 8TT, and A429 cJl'.pLlyeu ri contlrIlJOll',ly IIM dpfpI dl IrHI r,lIl' (3-6 fras:; pelletsjlnsec..tjday) dS c..ornpdrr.:d tu the (ontrol IfI',p(I', (JO J4 frass pellets/lnsectjday) rJurlflg th(~ 5 ddY') (Jf f'/rJ()'.,llrf' Ir) unlr/'dl"lj 111/·1 ( F1 g. 8). St raI n A3 1 J f! 1 1 ( 1 t (: (J f rd 'J S pro d Il ( t 1 Url ((J fTI PcH d Il 1 /, 1 () 1 Il (l (/ JI il r IJ 1 • 55 • 1 1 • • 56 A 110 A30 • A429 100 ~----~ : 8TT 90 b A311 .... 80 ~ï 0 ----2 CD en 70 c::: 60 .... 50 ~ "CD 40 Y --t------=:! , G) 30 o..~ ~ ~~ en 0 20 ~ !: 10 L- c::: .... 0 a .... 0 o .... a 50 100 150 200 250 300 350 0 L- Dose (J.Lg proteinjml) CD ~ -00.-; E • ::l B c 11 0 A30 •~ A429 c 100 ~ 8TT .- 0 A311 CD 90 en c 80 r=O.97 CD Lo. 0 70 CD 0 60 50 40 r=O.97 30 r=O.96 r=O.92 20 10 l 0 1 .75 2.00 2.25 2.50 2.75 • Log dose • f IUlIn.: 5. fhe relatlOnshlp between FP so values (dose inducing 50% decrease ln frass productlon) of Baci71us thuringlensls strains A30, A429, BTT, and A311 and their correspondlng LDsovalues (median lethal dose). • • 58 • 1400 1200 ...... E 1000 ...... -c ....CD 800 0 ~ a. 0) 600 • ~ '-'" 0 400 Lt) A429 a.. La.. 200 r= 0.99 \ BTT A30 0 ---1 0 100 200 300 400 500 LOSO (Jl.9 protein/ml) • • II • • 60 • >- .g 1 6 ...... • A30 en v A429 Ü 14 ~ 8TT Q) en o A311 .~ 1 2 • CONTROL o .- ...... 10 en +- CD 8 • CD a. en 6 en o ~ '+- 4 '+o ... 2 CD oC E :l Z 0~~~--~~4--~5--S-o 1 2 ~ Time (deys) • • Ilqure 7. Cumulative 7-day mortality of the adults of Listronotus orcgoncnsfs thdt had survlved a prior 8-day exposure ta the LO~~ dosage lnsectlcldal crystal proteins of different strains of B. thurlnglensls . • • 62 • ...J 0 a::..... z 0 u...... m CJ) N '"'It -< 0 .,.... fi) ..... c: -< 0- 0 CJ) • e L- '"'It (f) -< - ..- .,.... fit) -< CJ) CJ) N -II( e fit) -< o 0 0 0 0 0 a CD li) ..q- tt') N "'"'" • • ~ l exposed to LD~G values of 8aci77us thuringiensls strains A30, A429, Br 1, and A311 • • 64 • ..... • A30 u 14 v A429 CD T 8TT ln ~ c ...... l T 0 A311 .- 12 f"""'-- ...... CONTROL ".....ln '-1'" . -CD 10 l -2_~-~~l -CD a. ln ln ~ 0 ....."- ..... 0 • "- CD ..Q E ::l Z 1 2 3 4 5 6 lime (deys) • Detection of B. thuringlensis-like bacteria • B.thurJnglens7s subsp. tenebnonls readlly grew on NAP whereas no (Jrdfll neÇJatlvP bactf:rld grew c.onflrmlnÇJ the validity of the medlum for "plf!(tlnq for lJ thunng 1 ens/s (Table 2). B. thunng/ens/s-like organisms vJ(>rr' dpLpctpd ln and on dll structurflS excepL the haemolymph of adults, lilrv,l! CJut ilnd on the carrot on NAP mechum. In addltlOn, therr was no l!vldpIlCl; of B, thunnglcnsis-llke organlsms ln larval gut, haemolymph of larVde and adult~, elytra, and carrots when streaked on NA. DISCUSSION IhE' dl~CI,V(lry of B. thuring/ensis isalates with taxie activlty dq,tln',t colpoptoran'; (Kneg et al., 1983; Herrnstadt et al., 1986) has InJt latpd an lnt(!rpst ln evaluatlng lep toxicity and identifYlng and l'xpldITllr1 1',ol.1tp', 111< ludln~J ttlP (olorado pOtdto beetle, Lept/notarsa dcccm7/neata, • thl\ l'l11l I(>df hppt Il', Pyrrha7ta lut(lola, the boll weevil, Anthomonus ql,lncl/" 9I'Jnd,,>, thp ypllmIIlH:Jlwot'm, TL'nobno mo17tor, and the> black Vlne WII(\VII, Of /orhYflchu:, sulcùfus, (HerrnstdlH et al., 1986). lhlS study l'pPOI'I" Ill<' Il'lat IV(' tOXlClty of ~)everal stralns of B, thunnglcns/s wlth knowli .le t IVlty d ',I.\ Ihdl thl'l'l' Ollt of the ~e>ven stralns tested are pathogenic. to these 111',\\1 t" lhll Lll~ pl ,IYllILd)ll Jty of the natural diet (fresh follage) of the ,HIIIlt w('('vIl" Yl'Jl' l'OU nu has lead to the search for a more practical dltl'lllLlt IV!.', flH' pretrrred food source ln terllls of fecal productIon (as d IIlPd',Ut'\' of COI1:'U1l1ptlon rate) was found ta be the seml-artiflclal diet 1 (111l!1w,l'd of dlOpped folliHll' wlth 4~c agar. The fol iage can be chopped and "tnll'e! ~vJth w,ltpr dt ?O°C until reeded Members of the Umbelllferae 1,11111 Iv dl'l' ,1l1111ldtll dup to the presence of essential oils and reslns • 66 • • • Table 2. DetectIon of Bac177us thurlng1ens7s-llke bacteria ln/on lnsect structures of untreated adu1t L1stronotus oregonens7s and ln the insect envlronment. ~------~-~----~~~~-~~-=-=-~~--~~~~--~~-~~~ =====-~~-----~==-=~===-==-~~------~-~~------ TREAT~lENT BTT L At~F AF SJ Haem Elyt 5011 Carr ~1E[1ILI~l ------ NAP -'-+ + + + +++ NA +-'- ++ + +++ ~----~._------=-~-=--~------=--~--~-==-==~~=~=~------~=--=----=~------ L.lar\al gut; ANF. ~onfejadult gut. ~F.fed adult gut; 5J. gut of fed adults from another locatIon lSt. Jean-s~r-Rlchelleu);~aen.haemoly~ph form adults: Elyt. elytra: Carro carrot: BTT, pure culture of 6ac'71us tt~r7~g:enslSsubsp. tenebr70n75 (a pOSItIve control): N~P.nutrlent agar· Polymy~lnB sulfate _ ~~rlClllln;N~.nutrlent agar. -. presercE of B thur7nglens1s-11ke bacterla; -. absence. (Berenbaum, 1990). thoPPlng up the foliage may have released the chemirals cau~lng a ~tronq attractIon stImulus to the in5ect relative to the intact • f r f! ~ h f () 1 1 il q f! t h U', cre d tIn 9 a h 1 9 h con su mp t 10 n rat e . The a 9 M' C l e a r l y dope, not pldY ct fllltrltH;nal role but may help pn?sprve the texture of the folldqt: and i1llnlrTll/f~ wdlpr 10',:' fram the dlet WhlCh collectlVely would be COlidUClVf' tü feedlnq dnrJ/or frass prOductlOn The (het a150 ensured ITIt.lXlmUm InItIai ff'edlll(j of tO/lfl-contdrTllnateu dlet. AIU)(Jlj(Jh ri ml'lhorJ ln ',tdndardllp Hl(; potency (lU/mg) of thp dctlve IrlCjYl!dH!nt of EJ thllrlnfjl(!n';I~ ()txaln~ texlc ta coleopterans US1:1g the ColOY'ddo poLllo tlf'f!tlp dr, the.- tpst InSf'Ct has been proposed (Ferro and Gplf'rntel, 1939), d PUtJI1Cly avallable R. trunng7ensis-coleopteran ',Pf:C11lC ',!drlddr'l ha', not bepn dt'vE'loprd tn ddle (Tornpklns et al., 1990). Ihflrefor!:, the ,t( t lVP Inqrrdl(:nt ln the bloassay:, ln thl,) study was based orl ttH' pro!PIIl contl:nt of the cry<,tals per wright of spore-crystal mlxture () f (> de Il ,,! r ,1] Il ri', cl p t (: t'In HI Il cl bjan a 1y II cal nlP t ho d s . Ihp low ID.,! vdlu0" of ::,trdlns BTI, /\429, and A30 e~tabllshed lhese strclln" cl', the rnw,t tOXH. III adrlltlon, 811 has the lowest crystal protein ( 0 n ! Pli t f Cl l l OVIf~ d Il Y 1\4? 9. Th l C, su 9 9 est ~ th a t Bir p () s se <; sr s the m0 s t pot e n t toxln wlth M?9 III 'd'rond place Although the protein content (jlq/mg • ,>pon!-cry~tal Hllxturp) of c;tralns A409 anej 1'\299 are 10\>/ compared w1th the lOXH <,trclHI A30, then' corrpsponrllng LO ..!: values are relatlv21y high, IlllplyltHJ that ttH' rry',tals drf' lf~ss tone. As Judged by ltS LO~( value, ~lrJHI A30 Wd" found to be' relatlvely tOXIC, although lts .:.rystal p)"Oleln . (' 0 n t (' Tl t W,l '. 1] Hl li p r t li an t h il t 0 f BTT and A4 2 9 1 h l S S li 9 SI est s t h a t t. h e tClXH Ity of the pndotoxlll of straln A30 1'; less th" tnat of the latter t wo "t r a III S Thl' r il Il 1-- lTlCl 0 rd pro f t Cl)' 1C 1 t Y (J f the th r r è ' l e c t J d s t ra l n sis a funct lOTI of thr r,ercentage of ln~crt kill uelTlg SOUljrlt. The steepness of the slapf'c, WIll dlrtatc thl' rank of the ~tralns, e.g., thE' rank arder of tOX1Clty fOI' a r)o~" klll lS A429 > 8TT > A30 whlle for a 70% k1l1 it is RTl .., 1\30 ·1\42g ln illl (JSl",. thl' sl01'('~' wen' slgnlflcantly greater than or ,",IJill to llrl1t) thu'" lnscct dcm1sP was the mOl'e Immediate result of tJX1(O'I~ dS orr0"od to srptlcemla (Bucher.1973). Wh Ill' 11101 t J 11 ty of :-.urv 1vors pl'ev 1DUS ly exposed to lep doc;ages at ttll' ln'l' v 8TT were 5igniflC'antly diffel'ent from the cnntrol ~ll'()lIp'). but Ilot tnllll • each othe!', ThIs further sUYSll'sts that the lùttl'r Stl't111l\ dl(' 111lll'1' t(J:(ll than the formel' and th,lt the l'ffl'ct of <;tralns 1\4?Q dnd 1111 dt th,lt dow 15 IrreversIble. The lonS]-tenn Illot'tallty l'ffL'ct of thl''>l' "trdlll" LD~ûdose mqht be of l'lOnOl1lle Ill1pllrtanCl' lInde)' fIeld cundltlon'> ~1111l' lIllly a minimllm numbe'( of ':.pl'dyS wauld be rC'qllll'ed tn JchlPv(' l'ffl'lllvt' rOlltl'ol In contras:t, a more t'epet Jt Ive sprayln~1 C,Chl'l11l' rnl~lhl bl' ln OIdpl' 101 strain A30 ta obtain ~ll11llar n'sults. An alternatIve procedure for asses<;lrq lep toxiClty ,1<1<1111',t lll'pt I"', clearly establlshf:d ln this stlldy is the frds~ pt'OdUC\IOIl ,\',",IY, ,Ill IndIcatIon of feedlll~l Illlllbltlon lhe do<,e-re')I)(HI~l' ClJl'Vl''' !()) thl' effective strùlm re!Jdn!ln~1 ttH' dpcrhlSP ln rlurnlH'1' of fldC,~, p('II('\'. PPI Insect cone1ated viP 1 1 wltl1 thosp of the mol'ta1lty hl()d~C,dY III lIf'fll'I',II. the frass product Ion CUI'V(;', t 1 dU' il r!()',d to rLduced feec.llnS}, r1orta111y of 11l'Je< t" lalJ~)l'd by ICP<, l'. qf'I1(·t.lily .,lllw being medlated by a serIes of (''{enV, ~1I( Il il~) ((")',d\ 1011 o! !('('d IlIq, development of ~eptl(l~lllil From UllS assay offr~rs iln (lxpedlPrtl lIif'dns of rc~llilb1y detprI1l111111 lsolùte tOX1Clty dgillw,t t)(~f'tlr,', ',Imlldl' to UIf' dC,')dY Pyot()f(ll of VrHl F rankenhuyzen and Gn nçjortr'rt (1991) I\t all test d()Sdqe~ of thp hlghly tOXIC ',lrùlfl'" j(lpdlnq l'lrI', slglllflcantly reduc.pd by the thJrd ddy of trhltfll(>nl. Br!yolld Ulrlt pf'r lod, feedH1':J t'Plllùln~,(j vpry IOvl Tttl~ mdy' Jrldlratr' Ihdt Ihf' quI vld', ',('vr'rpl,/ dél[,ld~Jf~d by ddY thr i '[' folloviJWJ Into,H,Jl10rl dnrj f>XPO'.tJr l ' 10 IJrI:rr·,tll'rI cJlr:t, ft'a',~ pt'OductlfJrl n'::JdlrlHj VI'f, IOvl f(n Irt',('(l', ('/rJo',{'d ln Iltl> effectIve :..tt'dlflS, l'lhllh ',uqqr.r,t', ttldt Irlr, (jIJI hdrJ Ilrtr]r'IIjOn r, Irrl"n'I',II!II' paralysls, Thl::; l', lmllin • UII' ::,pruCf' bud\>IIJrI:', Chon'.toflfJurd (UIII/fr'/tinri, \r) vlhld sublethal Ir:vr·L of [j thunngu.:fI',/', rJlrJ nol Hlhdlll mlrjrjlJt 11',',1)1' r e q p ne rat lOri (l,,, l ni' et al, 1990 ) The d0lE!cllfJn of U thurlngu.:n,)/'j-II'f r• bd( Ir'rld Irl thr IjIJl r,f • 69 IJntrr:atr:d ln',f:CtS from 2 distinct cultures made it impossible to fulfil Koch", po~.tulate u',lng the present method. The absence of these organisms • Irl thf' haemolymph of ttle insects ~uggests that the insects were not dISf:ilf,ed. lhpse mlc.roorgamsms mlght be part of the natural flora of the IW,flr!. ÇJut which was plcked up rrom the sail. Additional tests are rr:qllirpd to lonhrm thp ldentity of B. thuring1ensis-l ike bacteria. ln conclus Ion, 3 of the 7 strains are promising for use against the (arrot wflPvil. ror gr(ldtf~st efflcacy, an optimal relatlonship between the ill',(:c.t and thp toxln is rr:qlJlred. Therefore, further research into thC' 1TI0rJP of Je t lOn wou Id !Je llsrful . • • 70 REFERENCES • Bélair, G. and G. Boivin. 1985. SllSCE'rtlbil1ty of th€' carrot wüpvil (Coleoptera: CurclllionHlae) ta SUnnenlcma feltiac .• S. biblonls and Heterorhabdltis hel7othldls. J. Ncmatol. 17: 363366. Berenballm, M. R. 1990. [volullOn of spl'clal17dtlon in lnsrrl-umtwllifpt' assoClatlOns. 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Binding of the d p l t cl (' nd () t 0 X 1 n f r 0111 Ra C T 7 1u s t h uri n9 7 en sis t 0 b t'U S h -b 0 rd er me mb r an e veslClc', of thl' (ahbage butterfly (Pieris brasslcae). [ur. J. UToehcm 173' 85-91. Hoflllclnn. ( ,1\ VJlld('l'bl'lJll~Jen, H. Hofte, J. Van Rie, S. Jansens and H. Van MplLIf'I't. 1988h ~peclflcity of Baenlus thunng7ensis delta l'fl "ltl'::' 111 the t'lIsh-border membt'dne of target lnsect midguts. Proc. Nat' Acad. Sel USA 85. 7844-7848. !lultf', Il ,Incl H. R. Whllclpy. 1989. In~('ctllidal crystal protelns of I?acllius thUllf79ICf):,/s NIC1'ob701 Rev 53: 242-255. ~,r IPli. fi . fi. ~1. HUClet' drl(J \~ Schnpttet'. 1987. Bad7 7us thunnglensis var. tcnl'bllanlS ~t)'JJrl ~'-7 l~ ldentllal to the formerly in Get'many l~oL1tpd strJlll BI 256-82 R. t ssp. tcncbl'ion7s which is pathogenic tel roll'l1pt('t'JIl ln~ects. J. App7. Entama7. 104: 417-424 . ,,-, 1 ('li. V rI, A ~, HU9pr. C; A. Langenbruch and W. Schnetter. 1983. • 72 Baci71u5 thuringiensis var tenebrl0nis, a nf'W pathotypf' pffectivl' against larvae of Coleoptera. l. Angew. Entama 7 . 96: 500 508. • Kurstak, E. 1962. Donnees sur l 'epizoot Il' bacter ienne ndt un'Il (' prllvoquPl' par un Bac ill usd li t YP e BdC 1 1 1LI 5 t h U rI n9 lt:' n 5 1 ~ ~, II r ! p Ily s t l ,1 kuhniella Ze11pr. Entomoph,1ga MC'm. /lors Sel'. 2: ?45747 Knowles, B. H. and D. J. Elldr. 1986. ChëU"dCÎl'I'll purificatIon of a pldsma mCllll,rdrll' rerefJtol fOI' /iacl11Ll,~ thudngi'2nsis val". kurstakl lepHioptPrùn specl fic b PlldoloXl' . J. Ce17 Sei. 83' 89-10l. Lilly, M., R. Ruffel and H. Soumervi lle. 1980. Puriflcùt Ion of tht> inseeticldal toxln ln crystals of BaC7 7 7us thunng/ensls. lJ. Gen. Microbial. 118: ]-11. Martel, P., H. J. Svcc and C. R. Harri~. 1975. Mass rell!'lIHj of 1Ill' (MTot weevll, /7stronotus oregoncnsls (Coleoptrra; Cut'lllIIO!ll controlled envlronfl1pntal condit lon<,. Cano [ntomal 107. <)~ 98. Milne, R., A. 7. Ge, D. R1VeY'S and D. Il [)edn. 1990 \pl'(lf](lty (Jf insectlcldal cry~tal protelnc,. ln. IÎnalyt ICdl rtH'IlII',Lr'y of UaclllL1~ thuringlens7s, eds. L. A. H1Ckip dfld W. f lIdl. pp. 22-35. Washll1qton D.C : Amencan Chpnllc.al SOL1PLy. • Moser, L. 1990. 1mpPflal Chemlcal Indu<;trïes Hlolo~l1(al Product.~, North American Technlcal Centre, Ml2,~lS~alJga, Ontario, Canadd (c,pc Appendlx) . Pepper, B. G. 1942. The c.arrot weevll, Llstronotus oreganenc,n (Hohe) ln New Jersey and lts control. N. J. Agnc. Lxp. Str. /Juil. 693: 1 20. Pepper, B. B. and L. E. Hagmann. 1938. The canot wPPvll. 11~troflolu~ oregonens7s (Boh.), a new pest on (plcry. J.Lcon. fntomol. 31: ?62 256. Perron, J. P. 1971. Inseet pests of c.arrots ln Or~drlll \Oll~ of southwestern Ouébec wi th spec i al reference ta the carroI wp.ev Il, Listronotus oregonensls (Coleoptera; Lurrulionldae) [an.lntomal. 103: 1441-1446. POlnar, G. O., Jr. 1989. Examlnatlon of the neoaplectanld r;peC1P', fc/t/ac ~illpjev, carpocapsae Welser and blbionls Rovwrt (lJpm;d.odd. Rhabditida). Rev. Nematol. 12 375. POlnar, G. O., Jr. 1990. Taxonomy and biology of Stelnern(~nlatldae and • 73 Hr!terorhJbdltidae. In: Entomopthogemc Nematodes in Biological • Control':., ed':.. R. Gaugler and H. K. Kaya. pp. 23-61. Boston: CRe I lhp',i:., Hlltqen Unlver:'lty. r~ew BrlJnswlck, New Jersey. 141 pp. )dleh, ~. M., H r. Hdrns and O. N. Allen. 1969. ~1ethod for determlnlng Bacll/us thunnguYnsls var. thunng/ensis BerlIner ln SOll. Cano J. M/croblol 15 1101 1104 "1fl11rnoff, H. fi. 1962. fi stdining rnethod for dlfferentiatlng spores, cry')tal~>, and u':]]j of Bac/77us thunngiensis (Berlwer). J. Insect. Patho/. 4. 384-386. \orJ](>rvll]p, li J., Y. lamada and E. M. Dml. 1970. Lethal effect of plJnflPd ,>pOt'(l thurlnglcf1s/s on sl~vrral lepldopteran insects. J. Invertebr. Pathol. 16: 24J -248. "t l'V(>Il\OIl, fi B. 1976 ~easona l hl s tory of the carrot weev il, L / st ronotus orcgonen)/'s (Co1eoptera: Curcul ionidae) ln the Holland Marsh, Ontat'lo. Proc. Entomol Soc. Ontano 107: 71-78. • IOlllpklllS, G., IL fngl;.11', ~1 Mondelsohn and P. Hutton. 1990. HlstOrlcal il\PpctS of the' qUùtltlfl(atlon of the actIve IngredIent percentage for BaC/llus thu/ïng/cns/s products. In: Analytical Chemistry of fiacJllus thurlng/cns/:" pds L. A Hlckle and ~~ L. Fitch. Amencan (llPm](dl SOCll'ty, WashIngton D.C. pp.9-13. VolTI ~l',lnkpllhlly/t)ll, K ,Ind J. L Gnngol'ten. 1991 Frass fallure and pllpatlon fdllul'P as quantal measurernents of Bac/llus thunnglensis tOXH lty ta lppldoptera. J. lnvertebr. Patho7. 58: 465-467. \~lJlt(()lllh, \~ D. 1965 lh(l canot weevl1 ln Massachusetts. B1010gy and cOl1tl'ol. Unlvcl's/ty Nassachusetts Agne. Exp. Str. Bu77. 550: 1-30. • 74 • CONNECTING STATEMENT In Chapt0Y' 2, ;even strains of BacIllus thunngiensls aet IVl' ,Iq,lln'.\ Coleopiera were evaluated for Ulelr toXiClty agùlll')t tlH.' adul\ '.LI~ll' of the carrot weevll, Llstronotus orcgonensÎ:>. Thn.... c ~ll'aln~ Wl'l'(\ ~l'll'l'h'd with high toXiClty and one strain wlth low tOX1LlIy 10 drtpY'1ll1rH' d 1 f fer e ne es i n ni 0 des 0 f t 0 xIe 1 t Y 0 f t h(1 s t l'a 1n ~ 1 n Ch d PII \l';' 1Il l \ solubility of the crystals of the thl'er toxie str~l1ns comparpd wdh " weakly tOX1C stl'ain and the proteolytic modifIcatIOn of tll(' ~)()ll1ll]lll{'d crystals in carrot weevil gut extract were studied. • • 75 • Chapter 3 Solubilizatlon and proteolytic processing of Bacil1us thuringiensis 6- endotoxln in the midgut flulds of the car rot weevil, Listronotus oregoncnsls (Coleoptera: Curculionidae), in vitro • • ABSTRACT • Several stralns of B. thuring/ens/s active agJlnst Colpoptl'I'.l WL'rl' seleeted for tOXiClty agalnst the adult carl'ot WPl'VII Both. solubilization and proteOlytlc processll1q of the ,\-endotoxln~ of thl' tllXll strains (8TT. A30 and A429) and a weakly taxIe <;tI'<1ln (1\311) \oJl'l'l' (\I(,lllllf1l'd to determlne their relationship to toxicity. Crystal ~olllhilJty IIHI'l\.1''\\(\ as the buffer pH lncn?Jsed and. wlth the eXel'ptlon of ~trJln I\JII, W.l'. correlated wlth LO"" values lmplylng that tuxin availdblllty IIldy PXpLlll1 toxicity. Proteolytlc ,lctlvatHlIl of the lTy',Ltl:, f\'ol11 tht.' tOXH h,lctl'l l,II 1so1aLes with carrot wepvll qut extract YH:lded 51ml1,w l'Il't tI'OphOl'l't 1t profIles. Unllke straln A311, strall1:, 8TI. A30 and 1\429 PO',\(","l'd d putatIve toxle proteln band (66-67 kOa). Thesp rl'stilt" <,1l9 relative lm·/ tOXIClty of stranl A311 crystal 1<; dup, ln p,1rl, 10 tlll\ a b 5 e n c e 0 f the t 0xIe mOl et yIn the Il f' nd () t 0 X 1n . • • 77 INTRODUCTION • The Cdrrot \'Ieevll, Listronotus oregonensis (Le Conte) (Coleoptera: ~JlJrc.ullonidae) is d major pest of Umbell iferous plants in the northeastern ',trl!J!'. of the UnIted ~tate~ of Amerlca (Chandler, 1926; Pepper, 1942; WhltcornlJ, 1%5; Grclflw. dnd Otto, 1979) dnd CdnarJa (Perron, 1971). Its Il rd f! r r fi d h () ':. lis (d r rot S, ,Hl d wh r n t h (l HI ') e c t l S l e ft un con t roll e d, u P t 0 4ü'/" of t hp (t'op hdS beprJ repor tpd to be damdged ln Quebec (bolvln, 1985). Ih(l ddllit fpnldl~~ lays e~lgs ln lhl? petIole and crown of the carrot and, Ilpon prnf!nj!'rJ(p, the' larVJfl tunnel down lnto the sOll and pupate (Perron, 1971). Itlf' (drn)t Wf!(!vIIs overwlnter ln the so11 as adults. Three highly viruiplil strdlns of Bac/llus thunnri1ensls active agalnst the adult stage of ttlf> IJaclilus thuflnglCnQS 15 cl Gram-pO':ltlve, spare-formln'J, so11- dwpllllllj hMtprllllli (hdr'actPr1ud by lb db1l1ty ta produce lnsectlcldcll ( t' Y'> LII 1 1 Il (l Pr () t (l 1 Il " (J C P<;) dl j rl ne) "p a ru lat ion. The Ieps, also deslqnated (t'Y',tdl 1 11](' protoxlnc.; that dl~:)()lve 111 the rnlG'Jut have relatlve molecular • llld\',!", (Mr) of 10 klld-135 kDc1 dependlng on b~ct(>nal Vdt'lety (L1, et cl 1 .. 199 1 ) (, lit Pr () 1(' d l, (". )' p l e J <, eth (l il C t l ve t ü XHl wh 1 ch the n h l n d s t 0 t'P(Pptnt". on thp fll1(jqllt l'Plthpllum (Hoflllc1nn et al., 1988a,b) and induces 11',lk,\(jf' (11.111111' l" III t h(' illldqut Cf! 11 rnpmbranes (Crdwford and Harvey, 1988). Ill'dlh (JI th!' IIISl'l t pW.lI(,', due to ~Iut and hJcmolyrnph 1011 contamination Ipcldltlq dlt'pct Iv dnd lncll~'ectly tn starvatlOll dnd septieemlil (Hofte and ~Jhllplcy. 1989) rhl' bd'.,lS for the different1al tOX1Clty of the B thunng7ensls '.tldIW, to)' tlH' canot weevll may be attl'lbuted ta one of the three stages d'.'.(}, lcltl'd wJth thl' mode of JetlOn of the c',-endotoxln for other lnsect .pt'l](l'. cl'> ptop()\l'd hy At'Oll~OIl et al.(1991). These stages lnclude . Ulllhlll.',lll(111 of Irp~. Pl'ott'olyt le det Ivat Ion of the protoxin and to;.nn tlll1dlJhl 1\) "Pt', die t'l',-eptot's The Importance of solublllty has been dt'llll1l1' 11 ,lfed ftH' 14 Sl't'otypC' nf B thunnglensls stt'alns agalnst three Il'~'ld')f1tl'r,ll1 :,pec,l'S known to respond dlffprently to the ,I-endotoxlns \J,lqUl't t't al .. 1987). I\lthough pr'oteolytlc processlng of the protoxin is • 78 essential for the act 1 vat 10n of the toxi n. rapld deg adat ion of tlH' toxin ml ght be an addltional factor expldlnlng rpd uced tnxlclty. HOWl'Vl'I' • • accord i n9 to Slaney et al. (1992) li S lI1 the rapid pi'otcolyllC degl'ùdatlon of th!' tOX11l 111 dlqe~tlV(, f JUld of tIlt' 'former in'.cct dl0 not hlndol' tOX1Clty JI\l' toxlcity ,1Ild <"pl'clf H ltv of " endotoxlns have been :,hOWIl to correldte wlth tlw IJrP'd'rHt' of tl1qh ,dt Indy blndlTlCd sltes ln the brush border nH'n1l1r,lIlp of Lln'pt lWd'( t 11l1dqut eplthellum (Hofmann et al, 19SBù, h) Thpll' c;tlldy (()f)(!'llti'.lIt'd un Il thuringlen,'s ',traIn" WhlCh dlltered ln thl'11 1f]C,l'ctH HLll ,\{ t IvlIy towdrd tobacco horrlwol m. Nanduca sex ta, dnd (ùbbd Receptor blndlng stllllle~ by Vdll HIe ct dl ( 1990 ) resistance of thp 1 abol'JtorY-:"f'lect!'d l nd l ,Hll1ll'tl 1 molli, PI od 1 cl interpunctclla, straln tu [J thUrJnglcn~ls \ub'.p. berl/Il/'/' Ill' W,I'> correlated wlth a redllctl0n ln dfflnlty of ftl!' 111f'l11ht\ltH' t'p\l'pl())' flll tll!' tOX1C protpHl. Howevt:r. dlflrf('llt CldS',!", of lCP IIldy blnd to dl',t Hl< t receptor Sltl:S (Hofrnann t!t al. 1988b) Thl:, rnay (lXpL1HI Uw '.(l!1·,ltlvlly of P. Interpuncte7 la to il ~e(.oncl typP of ltl')(lclH Id.!1 t IY'lt,lI prOlf'lT1 111,11 • seems ta rec09rllze a dlff(lrl'nt recf'ptor (Vdrl I~II.' 1'1 JI. ,1(jlJO). The examlnat lon {Jt Ur: thrpp steps If! lhp modp of J,( t Ion of IL thunnglensis ,1 -pndotoxlns should dptennln(! the fa! tOI', rf",port"lblr' for the differentlal acllvlty of the four stralns of Il thunIlCjU?fI)/', ù~ldlll,>t the carr"'ot weevl1, In thlS study, the two pnfl1clry k!~y '.fflp'" 'loJutl1llty and proleolyllc processlng. H1Volved Hl the aCllVJtlOf] of thl' tO;l1fl wpn ' lnvestlgatecl. MATERIALS AND METHODS Chemlcals ManobaSlc and dlbasic potasslum phosphate (KH;'pO" and K;fiPO/ .. respectlvely), 2-b-mercaptoethanol, bane. aCld (H1BO J ), phor,phorlc dllfJ (H j P0 4 ), and acetlc aCld (CH)COOH) were purchased from SlCJma ChpmH.al • 79 Company (MI'JJOUrl, USA). Glycerol and sodium dodecyl sulfate (SOS) were purchaserJ frorn thf.: UnIted Stdtes Blochemlcal CorporatlOn (USA) and Tns • HU from Boehrinyer Mdnnhelm (Quebec, Canada). Bromophenol blue was dc.quired from !.llo-Rad (Ontario, Canada), dlethylether from BDH (USA), and nutr lent dqar from BBL (Maryland, USA). lnsects Carrot weevll available in laboratory culture and penodically ',upplpmcnted vllth carrot weevils from Agnculture Canada Research Statlon (jt ,ledn-sur Hic.helleu) were used to malntdln hybrid vigor. The lnsects wprp rf!dred on carrot', al cl temperature day:nlght cycle of 24: 16 oC with d 16 Il photoprriod (Martel et al., 1975). For additlonal detalls consult rhelptf!r 2 /Jacteria! strdlns ElaCi /lus thuring;'ensls subsp. tenebrioms and unidentified B. thunng/!:n'>1s strdlns coded A30, A429, and A311 with activityagalnst • ',~tronotus oregonensls (suppl1ed by Impenal ChemlCal Inrlustnes, Illolo(jllal Products, ~1Iss1ssau9a, Ontano) were grown on nutrient agar ',upplempnted wlth (:qulmolat' concentratIons (0.05M) of KH 2 P0 4 and K)1PO" (pH 1 0) Artel' 5 ddyS lnCllbatlon at 30 oc the bactenal lawn WhlCh, based lIpOll IIIIUoscoplr f'Xallllnatlon conslst p d of veç)etative cells, sporangia, r ',pOI'P", Jnd (rystd 1 S wa , th('11 <;cfdperl dnd lyophil ized. H,dgut ,solat ion and digest ive fluid pH TIll' auult carrot wecvlls wcre anaesthetlzed for 10 min with dll'thylethet' 111 J Jdt' If1la9()~ wlth amputated legs were plnned at the pnHwtllfll ,Ind Ll,t atldOllllnJ 1 ~l'~lll1pnt onto a wax pl ate. The al imentaY'y canal \>/d', (,xpll~.t'd hv,'ernllvulll tht' eiytra, hlnd wln9s, abdomInal tergltes, flll'LlIlOtUrll, .Ino plonntlllll (Cdlder-, 1989) Slnce protease actlvity is Illl dtl\d ln the f1Ild~lut, Ull' pH of the antenol' and postenor midgut was dt\tt')'1111nl'd ll',lflli l' f1llCt'ol>lpctrode (Beetl'odeT': pH nllcroelectrode, World • 80 Precision Instruments, Conn., USA) and d llqUld Junctlon n'fl'rPIHl' electrode (World PrecislC'n Instruments, Conn., USA) conllPctt'd ln tilt' pli • meter. To oetermlne whether the type of food lnfluC'ncl'o mld~lut pH, thl' adul ts were fed on elth01' fl'l'~h foliage 01' choppl'd fol iJ~ll' with 4"" "q.!\' (Chapter 2) for thrpc days pt'lfW to exposing tt1l' qut. TWl'nty pli 1 (',lJll1lJ'o were taken for ellch trelltment. So7ubi1ization of crystals at se7ccted pli values Ta study the cffeet of the pH on cl'ystal solubllilat ion, 1 111<1 of spore-crystal mixture of each straln was wùshpd 3 tlmllS wlth 0.01';,· lrltoll X-IOC (Chapter 2). The final pellPl Wù'> suspended ln 200,tl of UlllVPl",,11 buffer (2.7ml of con(entrated H,PO", 2.2911l1 concpnlt',ltpd q1.1 aCld. and 2,48~1 H,BO, pPt' 100rnl rllltll volulllp) ,li j.)H G,O tll 11.0 Witt! constant lonlC, strenqth (KollfT l't a7., 19(2) in 1.5rnl IItlcro(l'lItt'ifuqe tubes at 30 oc for 2 hrs. IÎt 30 111111 lntcrvcll~,. de',iqnc.ltpd ~)Ij l ,t! Iqllot,> 01 a.5M NaOH were added tu ,ntest prot{'oly~j<, (Hnlj)otJ... ,1Tld (lltll('I, 19<)0) The samp)es were centrifuged at 11,750 Xlj fur 3 mlTl dllll th,· ',UP('trld\.ITlt assayed for Its pr'ot r.'ln content dccordlnq lo th(~ f)t',ldlold flIfltlt()d (19/b) • USl ng a BIO Rad pt'ott'lrl d::'SdY "1 t (lllO Hdd, OnttltïO, Cdllddd). HIf' d',',dy l', based on the c.olout' c.hanqe uf iJ dy!! III rp',porl',P to ptotf'IJI hlndln!! Bov 1 ne serum albumln was used a .... d stand,mJ, Thf' pen l'Ill ')ollllllllLy Wd', ba::,ed on the arnOlJnt of proteln Irl the slIperndLJrlt rpldllvP to thf' tot.li solubility of the crystalsdlssolved ln 200 ILl of 3.3MNrtBt (BI'lllhdt'd, 1986) , Ta determlne the amollnt of proteln rwr crystal l)(!r :,trdlrl, thl' pellets were resuspended HI d knovlfl volume of Unlvenal bufll'Y dt UIf' correspondlng pH. dncJ th(· nUlTltwr of lln\olutJlll/f'd cry',tdl, COIHlt"rj !J',lnfj a haemocytometer. ihe absort)drl'f; of the (jyr:-prot p ln cumpir'/ (Jf 1iii' solubl11zed crystals at 595 nm Wd', thprl '.lIhtra r tf>(j from th,. to!.tl ab sor bil n cel n 3. 3 ~, nd [3 r ~. (j lu t Ion, 1ri t li (l 1d! t l' T ',!) 1Il t 1(J fi, th!, (r y', 1dl, v/et-e totall y solublI17f:d. TIlt: d lfferf'nrJ' ITI df,' f)rlJdrl' (> V/d', t(,HI',ffJrrnf·d to amount of prot':ln cotrr:~pondlng tri tri': un',fJIIJIJIII/p(j rry',t.dl'. (pg/crystal/straln) • 81 Residence time of food in the gut • To determlne a tlme range for the mldgut proteasl' actlvatlOn of the protoxlns, lt was necessal'y to ascertaln an apprOXllndt p rangp of t lIm' tll,lt the lep would be ln the gut Adult weevils WE'rp pnmarily fE'd C,llTOt lllnh (orange C010UI') for 3 days. They were then fpd folld~jl' \~1t11 4'" ,II/,l) (green colour). The tlmE:' from the lnltlatlon of lrl~('tt fl'l'dlll\l on tilt' green coloureu food source to the fll'"t Obc,f'rvdt 1011 nf ql Pl'Il f 1',1"" W,I\ determined to be 68-159 mln Whllp recogrllllnq th the upper llmlt for incubat lng the crysLlls wJth lII1(jqut f IUH!" W,I" ? Il ThlS tlme 1S regarded val Id due to the mldqllt occ..upyinq dpproxllII,dply 75'1:. of the gut volume. Electrophoresis of protease-digested crystals The alimentary canal of adult weevlls wpre eXllsrd a'. dp')crdH~d prevlously ("Mldqut 1<,01at10n dnd dlgP~tlVP fluHI pH"), h0I110ql'rll/Pl! Hl 50mM Tns buffer (pH 8 0) dnd U1Prl centt'lfugl'd at Il,7S0 xq f()1 S IIIln • Percent salut)] l Jty of CI'v"tJI protl' Ill', of thf> strdlTl', ln ln', bIll fpl' ,II pli 8.0 was not sl~lnlflCJntly rldf(>rent trom thJt III Unlvpr',cll bufff'Y dt th" same pH. The cry(,tal~ won' solutJlllled a~ ln "',olubil17Jt lOti of (Y'Y"tdl', at selected pH valuc~,", but WItt! 50rn~1 IrIS hufff>r (rH 8 0) ,J', 0PJ!o',pr! tl) Unlversal buffer. Prellflllnary results of (Y'y"tJI ',oll!tJII Il ,il 1011 established the same level of solubllllatlon ln 50 mM ln', I>ufff'r (pH B.O) as ln the Urllver',al buffer al UH' :,anlp pH. Ihl' ILP ',OIIJllfJtI'. V/l'YI' r lncubated wlth qut JUlce , (1 5 v/v qut JUlCP:c..ry',tdl protplll ',(JIu! Ion) produclng a fInal c..oncpntratlon nf 311~J of cry"tdl protplrI !Jf>r 1 III of fInal suspenSlOn. 1hl' sample'., were lTlcub,Jted at 30 oC for 5 mlrl, 30 min, l h. and 2 h WhlCh span thp t1me of tood PdS"d(:!Hl(J in ttH' Sam pie" d l ~ S () 1 V f: d 1 n ',lJ l Il h 1 l 1 lIn fJ tJ Il f ft 1 r (0 St~ 1rI', 1\ Cl, p 1\ 6.8; 10% qlycerol, 10~~ w/v SOS, 5% 2-b-rn p rcaptof!thdrlol, 0.05'1" w/" bromophenol blue) (Laerllmll. 1970) ln d ratio of 1.3 wprf' boil{,rJ for S mlrl at 100 oC, loaded onta a sodlum rjorjecyl ',ulfatp-poIY COOHld',lP bllll' (BIo Rad, OntarlO, Cdnadd) ln fixatlve (40% methanol with 10% a C(~ t 1( tH 1 d) d nd ri C r, , il 1 n (? d i n the f i xa t ive. Data analysl~ Stallstlcal analysl" of palred data was performed uSlng Student's t- 1(>',1. Ont: Wdy dndlY:'I" of variance (ANOVA) comp1imented by Tukey's test Wei', U',pd 10 COTTlrdrf' qrOlJll', of nleans. Statistica1 significance was taken lit p RESULTS • pli mcasurement of midgut digestive fluids fed on selected diets lht> Jnt· t 101 MIU postpt'lOr nlldgut pH readlngs of insects fed either dlPI wpre not Sl 'Iffcct of pif on crystal solubilllation l hl' (rystJl \olubllitv of thr four lsolates of reps lncreased as the pli lllln't!\l'd frolll G.O to 11.0 (Flg. 1). Straln A30 had the greatest "nll/hllilv lot thl' pH t,Hlq,' tested whlle straln A311 had the least. The pt'tlPntdll{l "olllbllitv at cl C11vpn pH vaned with the bactenal straln. At thl' pH nf the Illldqut ( S 0), thp ordèt' of solubil1ty for ti-Je stralnS was A311 (b9",.) " l\Tl (55°,,) "1\429 (3rr) > A311 \!~:o). /\ l ot't l' 1 Jt Ion (t'-O gS) WJ) found hetween the solubi 11 ty of the more tOXll' leP l:,olate\ at pH 8.0 and theH LO,,, values (Flg. 2). LO=2 values IIlCI'l'd"l'd \'Iltl1 llllTE'dSlnq solublllty. TlllS was not the case for strain A311 whHh had J hlgh LD" value but a low percent solubility. • 83 • Table 1. pH measurements of the midgut of the carrût wccvil, listronotus oregonensis, fed selected diet..,. ------_.------._.------ MIDGUT SECTION d FOOD Anteri or Pos terior Fresh 8.19 ± 0.17 8.27 t 0.21 foliage Fol i age W l th 8.05 ± 0.18 7.74 ± 0.09 4% agar -- - -*" .. = ------.------_ ..... __ ...... - • a Mean ± standard error of the mean, N=20 • • Figure 1. The solubility of the crystals of different strains of Bac:'71us thuring; ens 1 sin Un i versa 1 buffer for 2 h at sel ected pH values. Each point represents the mean ± standard error, N=3, • • 8S • 120 A31 1 110 • A429 "• A30 100 0 8TT T- 90 >- 80 +- T/:/. .-- 70 .-.c /.-I ::J 60 • -0 /. g-2 (Il 50 O ~ 40 î---- / v.,/ v 30 ,/ \/-'\1 20 ".- t -- --/• 10 ___ e ~ 0 • 1 1 1 J 5 6 7 8 9 10 1 1 12 pH • • Figure 2. Relationship between the percent solubility of A429, 8TT and A30 strains of 8aci71us thuringiensis at pH 8.0 and their LO so values against the adult carrot weevils (see Chapter 1 for LO so values) . • • 87 • 80 o 70 A30 • 00 ~ 60 .... 0 • :!".- 50 .-.e ::J -0 r=O.98 (f) 40 • A429 ~ 30~/ 80 100 120 140 160 LOSO (J..L9 protein/ml) • The determ1natlon of the proteln content per crystal per straln revealed that thf~ least toxie stra1n, A311, had a slgniflcantly higher • protelfl content thdn thE" more tOX1C ICPs of A429. A30. and BTT (F1g. 3). flectrophoretlc profile of protedse-digested crystals in insect midgut fluids The major protein<, ln the lanes wlth dnd without gut solubll ized ICP,; for stralw) A30, A429, and BTT conslsted of polypeptldes with apparent molec.ular welght', of 70-71 kOa, 66 KDa and 33-36 kOa (rlg. 4). With ~trdln 1311, thf",f' polypeptides remdined stable after 2 h of prot!~olyt l( prO({~»JrI 1 h pO',! lncllIJdt lon cl', thi 66 kDa band lncreased These changps also ()(urrl'd wlth ',traln 1\42g ln ,ldrlltlon to the appearance of a th1n 58 kOa hand as 1fl( ubat Ion (ont !nupd <;mdller bands of 33-36 kOa also seerned to ',hlft allo '.n!,llIPI molecuLlr w(>lght polypept.1des for the h19h1y toxie lsolate'>. -Thf> lanjl\fJt polypept1de that appeared ln strain A311 was 35-36 klJd Hl Wl' Iqht when no prot(~ase had vpen added (Fig. 4). Prateolytlc • dl DISCUSSION Loleopterans lncluding t.hose susceptible ta B. thuringiensis subsp. tencbrl0nl~ hJVP been shown ta have an aCldic mldgut environment (Murdock et al , 1987, !<..ollpr et al., 1992) essential ta cyst.e1ne and aspartic prUlpd\p\ th.lt pt'f'dOnl1natl' Hl thp qut of phytophagou<, beetles. (The; and Housl'I11Jn, 1990) Howcvpr, tl1(' mldgut pH oi the adult carrot weevll proved to bp .,llqhtly alkallnl' I\lthouqh th1S nJ19ht. lmply t.h,lt B. thunngiensis ~trdln<, tOXll tn nlO<,t Loll'optl'ra wlth dClllll mld~lut fllJ1d would be lnl'ffettlvP dqdln~t the carrot weevil, purlf1ed CRY III gene product, the (r.Y~tdl pt'otPln of R thunnsnenSls subsp tenebrionis. wlth actlvity d~ld1nst Chrysomela scnpta (mldgut pH 5.0) lS soluble under bath moderJtely aCldlc and moderately alkal lne conditions (Bernhard, 1986; • 89 • Figure 3. Prote;n contEnt per crystal of the differcnt strains of Baci11us thuringiensis in Unlversal buffer (pH 8.0). Each bar repre~f'nts th!.' mean ± standard error, N=3. • • 90 • 8 7 ....c 6 ln :>..... 5 u "c 4- • ....CD 0 t... a. 3 en a. 2 1 0 A311 A429 A30 BTT Strains • • Figure 4. Proteolytic rroce~sin9 of the crystal.;; of lIacl/1U\ thL/l'lngll)f1',J~ strains A429 (A), 8TT (B), A30 (C), and A311 (0) III thl' quI .lUI( t' of the Llstronotus ol'egon()n'>1~. for <;trùlll<' A311, A30, ,md HI\, I,IIW 1 = gut f'xtract, l,lne 2 - ~oll1hlll/(~d (Ty',t,ll, 1,1111' ~ moleLul.lt'Wl'lqht <,tùnd,H'lL, Lln~l<, <17 quI l'XII.HI wllh Ily·,Lll'. ,II increa'>1rllj t lm!", uf lllcubùtlon (5 11111\, 30 Illln, h, ,Hld ? h) IIH' differen({~ wlth <,traIn 1\429 IS th,il Luw', 3 alld 4 arp llltl'ldl"n~,('d. Concentrat lon of protein applled h 30 Ilq/IO /,\. • • 92 • • • A B 200- -200 116- -116 97·5- -975 66- ---~ ..".. - --- -66 kDa 45- -45 kDa 31- ..... - -- -- , - 31 - ----~--. ~ ------...... 1 2 3 4 5 6 7 1 2 3 4 5 6 7 200- -200 116- -116 97·5- - -97·5 66- --.... - -66 45- -45 ~ 31- -- -,~- - -- .-- -31 21- -21 - ----...~ ...... -----~~ C D Koller et al, 1992) I11dh.lllU 1t potentlallv ,:-olublp IvJth tilt' !l1ldqul env1rolllllent of a w1dc r(\n~H' (lf coll'optl't'd!l ho"t\, ln tht' Pll",('111 "tudv, • the test ct~ystal~ trolll \tH' dlft{'t"pnt hactl'l'Ial ',(lU)'(t"" llll Illdlllli II1\' li thurlngicnsl <., \lIhsp ICf)['{111111l1 \ c(lntrlll \ 1IItlll"!.', ,lppl'dl'pd tl' 1111 11'.\"1'111 solllbillly thl'ouqhollt ttl!' f\H I.llhll:' t('~tpd 1111' )'pc,ult dlttl'l', tltllli ~,(lill'I et al (lCJ9?) III \~hld) 11(1)'lflpd ll'Y'!.II', h,IlIII!l11tpd ',ulllhlll!).11 pH I,ll to 9.0. Thp dtfft'I'I'IllP [lldY )1'1 ll'l! h,Ht(')'l,ll 1',oL\t!, ,llHI Il) Ildtw,11 rond 1 t lon·,. llowP VP\O, t Ilf' (ont 11111011', lll\ 1'\'.1',1' III '0 luh 1 Il .'l'd l') illl' III III ,1 V represrllt 10d('hlll~j of ',por!' P)'lrtPln~ OVt')' th!' l'Il 1',lIlljl' ot h II III q () At pH>lO.O thp uT,lal" IlIdY hdVP ',Olllhlll/l'd ('Xplclllllllii thl' ',11.111' Il''\' III til .. solubildy oh,prvdt 11111. Ihl', hl'llIq Ill(' (d'.I', ',pOIl' \ l'Y'.LII 1lII\IUII". dl'p not dc(.(\pldblp loI' ()'y"td l ',Ulllblll/"t WI! ',tUtllP" 111,1\ 1',1101 III '"IV III,d sporl' (ry',tdl ',<1I1IP1 (><, l'WU Id Ilf' IIIVdild l,II ,llldIY',I" ut dlt IVlly ',P4'lll.l slnce \JMqUC't rI dl (19B?) \'P!HII'tpd Ihdt Il!1 '.lI"lf'!,! ddf' III',I'I\'. toxill Jct1vity ',p('(trd Wd~, the' ',drill' wltll bot Ir plI! \ f 1(,11 (1 y .. t,tl', ,Itld '.pOII' u'ystal IlIlxtUIP'" Itrl' l'l",lIlt" dl.,o PO',I' Ihl' qUI".tlllll .1.10 whl'Ihl'l 1111' carrot w"cvIl bc'lor!q', tu tlrl' Iypl' III IJlOIIJ1 (Jf In',!'( t, Ih,t! 11'1111111' 1111111 spore~ and (rystdl', for 1I11(lXI(,1I101l \olllhllllY Id tlll' t(IY,III, Whlll' qen(Tdlly not thollqht 10 h(l d b,1l \'11'1 III t()fl( Il v III ',II ,(l'pt rllll' III',I'(! , • 1 S cl n p (, ',P 1d l il l '. t l' Il 1ri t tr f ' (' '{ Il 1 (",', 1() II () f 1(l! \( 1 t Y U' () 1 II' \ (' t ,il , 1 () q ?) Th (' (1 V'. \.1 l ' , () III li 1 1 1 1v (1 f '. t 1 Il 1 T1'. 1\4 ? q, fi H). ,III d Il Il, vi fi 1( Ir d 1 l' 1 l' l ,II 1 vI'I Y tOXll ta Hw t1dult (clno\ W('("v' 1 1 (t1l,qJfPI 7), dt pli BOl', ',I more ~01uIJ1(> thdll ttr,1I of Ihl' Il'',' !OX1< ',11.1111 1\111 f!rl'. '.llqql",t', t!rd! the rlI](I~lIt 1,'r\V1IOllITlPril 1T1dy hl' IT\()I!' «()t1dllC IVI'!I) '.Olllhi l/Irlq t III' tllX !fI'. (Jf the formel ',trcllll'~ ttldrl r\3 1 1 ICI' ':> 1n CP exp n: " " 1 () Il () f t 0 X 1 ( lt 1 l' d 1 Il Il ( t 1 (J tI IJ f ',II Il JI) 1 1 1 1 1 d t t III' nlldqut pli (8.0), (1TI(> 1'1011 Id l'l.pP(t 10 d',',(J(I,dp hlqh IO/I( Ily 1'111t! Irlqlr crystal sulublillv \111 pl 1'.lnqlv, d ypl,lt IVllly 1111'1 '.ollll)lllly l'l,l', lili PYvI'd for thl' mo')t IOXIC ',lrdITI (/\4?()) l'litt! lTI(IPd',lrrq ',()IIJhlllly ((JYll'ldtIUIj vJlth lncred'J IrlC) lI), vrllul", llil', lllyf (rystal sfJluhlllly rTllqlrl ',lJIJW",j 111,11 thr ,jlll'/l' III/ITI', I)f Ih, III(HI' t(J/11 s t r cl Hl " tJl Tl d \ f) r Pl': pt (J r " l'II t h q r i' ,i! " 1 d f f 1II l 'f d TI d / 1) r 1 rI ri 1)' l, (f' 1 1 IJ ! rit cJamagE: rnOrE2 qUlfkly th,lrl th" \1",', lU/Il l',oldl,·', The arnount of tutdl prut"lrl per rrr,lcJ! "'/d', rll:(.jdLlvr·lf 1(IYYI:ldlr·(j wlth the lnsectlC.Jddl dCt1Vlty of thf< ',trdln ',Incr' v/lUi Ihl' hllJhll t()/I( • 94 :.traln', A429, A30, dnd BTT, hav1ng the lower total proteln content per cryr,tdl th'lr1 the least tox1e stra1n A311. ThlS also suggests that • tOXlclty may be more dependent on the solub1l 1zation of specif1e proteln (()rnp()n(~nL Hl th" c.rystdl rather than the quantity of prote1n ln the cryr,ldl:.. Unllkf' ttlf: Lep1rJopteran-actlve crystal, CRY IA(e) Wh1Ch pyotpolytlfc.llly bn:aks down from the 135 kDa protoxin to a stable 55-68 k[)d lOX1fl (~lanpy ct al., 1992), none of the strains tested revealed a bdnd ltHqpr thdll 71-72 kOJ. Thr: CRY IlIA toxln lS syntheslzed as dn IHl',tdtJle 70-73 kOd protoHl, wh1ch 15 eleaved to th~l stable 66-67 kDa protplrl Hl /. deccm/lncata ((drroll et al., 1989, Slaney et al .. 1992). \t t d Irl', 1\30, 1\429, dnd Bll possessed both polypept Idc's, and aftr::r }-2 h of Ifl(lltldt Ion ln '11/1 ftllld, th!, 70-73 kDa banri rJlfTiln1Shed as the 66-67 kDa hdl1rJ Jrl(t'Pd',f'd lfl lnlprl',Jtv wlth prolonqed Incubatlon Not surpns1ngly, ',trdlfl 1\311 dpppat(lrj tû lJck the> 70-73 kDa and 66-67 kDa bands ln the ,!I1'>l'IlU' of qut fil/Id, suqqpst 1nÇJ that the relat lvely \'JPak tOX1Clty of the ·)tt',IHI IlI (Ndkdllllll\l l't al .. 1992) Pt'otc01YS1,) of LHY rIIA ln the dlgestlve flUld of th" ((l]otcldo Potato Ilpptlp caused thr corn[l10to degradat10n of thp 67 kOa p(llvp!'pll!lf' afll't' 16 mlll (Sldnpy et al, 1992) However, none of the ',tl,llll', wlth po]ypf'pt1de 66-67 kDa showed any Slqns of deto'.<1fleation ,lft'>1 ;' Il Ihl" ',lIqqt':,t~, th.1t the taxIe rno1ety 1.) stable (1 e protease 11 1 "'\\.1111) III !tH' qllt tl1rouqhout the re')ld(!ncp tlme of food The failure (lf thl\ Ôll b7 ~Il,l !Jand of stt'dln:. A30, A4?9 and BTT to be d1gested ln the qut fllJld of thl' Lal'tot \ 1\ 11.' V Ill\' W 11 Il t h(' a tll l l t Y t 0 dl q pst th il t pro te 111 rUl'thl'l' t'('sl'J)'ch lnvolvlng rrceptor blndlng studies should be 1IIldl)t'tJ~l'n t!\ l'l'vp,ll the pn?scncr of a slte of actlon (receptor blnding '.111') nf tIlt' toxln'. ln Hw Tllldqut eplthel1um brush-border membrane of the • 95 carrot weevil. A comparative receptor binding study for thp 4 strain\ would be an important step in further understandlng the mode of action of • the toxic strains, and in examlnlng toxicity of strain A311. • • 96 REFERENCES • Aronson, A. J., E.- S. Han, W. McGaughey and O. Johnson. 1991. The solubility of InclusIon protelns from Baci77us thuringiensis is dependent upon protoxln composit1on and 15 a factor in toxicity to lnsects App. Fnv. Mlcrobl0l. 57(4) 981-986. Bernhard, K. 1986 Stud1(~s on the delta-endotoxin of Baci71us thunnglens/s var. tenebrlon15. FCMS M1crob1ol. Lett. 33: 261-266. Baivin, G. 1985. EvaluatIon of monltoring techniques for the carrot weevll, 11stronotus orcgonensis (Coleopterai Curculionidae). Cano Entomol 117 927-933. Bradford, M 1976. A rdpld and sensitive method for the quantificatlon of nllcroqrarn qlldntltll''' of protein utilizing the principle of protein dye tllndw Brussock, ~. M and 1 C. lurrl l'r. 1990. Use of sod1Um dodecyl sulfate- polyacrilallllde <..je 1 ell'ctraphoresis to quant ify Baci77us thunnglcnsls b-endotoxlns. In: Analytical Chemistry of Baci77us thunnglcnslS, pd". L. A. Hlckle and W. L. Fitch. pp. 78-87. WJShln~lton D.L Arnl'rlcan Chemlcal SocIety. • Calder, A A 1989 The allmentary canal and nervous system of CUrCl/llOnaldae (Coleoptera). gross morphology and systematic slgnlflcance J. Natul'a7 Iflstory. 23: 1205-1265 Carroll, J., J. Ll and D. J. fllar. 1989. Proteolytlc processing of a coleoptpran speclflc delta-endotoxln produced by Baci77us thurlnglensl~ var. tenebrIon/s. 81vchem. J. 261: 99-106. Chandler, S. C 1926. The econormc lmportance of the carrot weevil in 1111n01) J. Ecan Fntomal. 19: 490-494. Crdwford, 0 N and W R HJrvey. 1988 Banum and calclum black Baci7lus thunnglcns15 ~IJbsprCles kurstaki b-endotoxin inhibition of potasslUIll currE'nt across lsnlated mldgut of larval Manduca sexta. J. exp. Bl07 137: 277 286. Graflus, E. and M. Otto. 1979. Detectlon and control of the carrot weevil. MIchIgan State UnIversity. [xt. Bul,. [-890. 2p. Hofmann, C., P. Luthy, R. Hutter and V. Pllska. 19B8a. Binding of the <1plta-endotoXlTl from Baci1lus thunngiensis to brush-border membrane • • 97 vesicles of the cabbage butterfly. (Pieris brassicae). Eur. J . Biochem. 173: 85-91. • Hefmann, C., H. Vanderbruggen. H. Hafte, J. Van Rle, S. Janspns and H. Van Mellaert. 1988b Speclflcity of Bac/llus thurlngiensls ~-endotoxlns i seo r rel a t e d Wl t h the pre sen c e 0 f h l 9 h a ff 1n Jt y b 111 d 1 Il q ~ 1 t e ~ 1 n the ruçh-border rilemhrallo of target 1nsect Il1ldqut S. Pt'oc. Nat 1. Acad. SCI USA 85: 7844-7848. Hofte, H. and H R WhlU:ley 1989. Insecticidal crysLll prott'lns of Bacillus thurlnglcnsls. Mlcroblol. Rev. 53: 242-255. Keller, C. N., L. S Baller and R. M. Holllngworth. 199? Chdractf't'lZatlOn of the pH-medlated solubility of 8ac1l1us thurin9/(~ns,s Vdr·. san diego native t',-endotoxln crystals. Bioehem. Hl0phys. Res. Comm. 184: 692-699. Jaquet, F., R. Hutter and P. Luthy. 1987. Spe thur1ng1(~nSlS delta-endotoxin. Appl. EnvIron. Mlcrobiol 53: 500- 504. Laemmll, U. K. 1970. Cleavage of structural proteln" durillSI thl' Martel, P., H. J. Svec and C R Harll:'. 1975 Mas" redrtn~ of li]!' Ldtï,()t weevil, Llstronotus orcgonensls (loleoptera. CurCUl10rllddl') unr!I'Y controlled envlronmental condltHHIS. Cano Lntomo7. 107 <)!) 98. Murdock, L. L., G L. Brookhart, P. r. Dunn, D. r. fOdrd, ). I Cysteine digestIve protelnases Irl Loleoptera. Comp. !J1ochem. Physio7. 878. 783-787 Nakamura, K., R. Mural-tJl',hloka, M. ShImIzu, 1<. O',hlf>, K. MlklLufll, 1< Oeda and H. Ohkawd. 1992. In::.eetlcHJdl dctivlty and prou",',lrl Pepper, B. B. 1942. The carrot weevl1, Llstronotus orcgoncnJ/s (Bohf!) Irl New Jersey and lts control. N. J. Agne. [xp. Str. Bull. 693' 1-20. • 98 Perron, J. P. 1971. Insect pests of carrots in organic soi1s of southwestern Québec 'foIi th spec 1 al reference ta the carrot weevil, • Llstronotus oregoncnsls (Coleoptera: Curcu110nidae). Cano EntamaI. 103: 1441-1446. Slan(!y, A. C., H. L. RobIns and L. Engllsh. 1992. Mode of action of Bacilius thurlnglensls toxin Cry IlIA: An analysis of toxicity in 'cpt lnotarsa deccmllncata (Say) and Olabrot Ica undecimpunctata h Qlv a rd 1 El a r ber 1 n sec t BIO che m. f1 ole c. BIO 7. 22' 9 -18 . Thel, tL r~. and J. G. Houseman. 1990. IdentIfIcatIon of cathepsln B, 0 and If ln the larval nndgut of Colorado potato beetle, Lept7notarsa dcecml1ncata Say (Coleptera' Chrysomrlldae). Inseet Biochem. 20: 313-318. Van fhe, J., W. Ii. McGaughey, D. E. Johnson, B. D. Barnett and H. Van r~ Q lIa e r t. 1990 . MQ cha n 1 sm 0 fin sec t r e sis tan cet 0 the mie rob i a 1 InsectiCIde 8ac1l1us thJnnglensls. SCIence 247: 72-74. Whltcomb, W. U. 1965. The carrot weevll in r·1assachusetts. Bio10gy and • control. UnIversIty Nassaehusctts. Agne. Exp. Str. Bu71. 550: 1-30 . • 99 CONCLUSION AND FUTURE RESEARCH • Laboratory studies lndicated that srlcctpd str'ains of Raclllus thuringiens7s active agalnst Colroptol'il JI'l' potentlLllly effectIve bialogical control agents of the adult carrot wl'pvll Th(>~p ,>tr,lln~ could be Incorparated Into an Int('~JT'Jtcd pest mdnJ~J('II1l'nt (IPM) PI'()~P',lll1 Bdc711us thul'lng7cns/s subsp tenebnon/s and ,>t)'Lllll~, 1\30 ,111<1 1\429 were selected as the most taxIe ,lmonS} ttll' ~pVl'n <;tl'dln<. th,d WI'I'l' t('\tpd agal n s t the c. a rr 0 t we l' vIl il du 1t ~; Th l' fa d th ,\ t <; u r v 1 v 0 r '> () f t tll' 1Il "l' tt havlng been exposed to thr"c threp strcllns do Ilot )'L'SlIllIt' llo)'lIldl lt'vl'l., 01 feedlng suggests that Irt'pvenlble gut pal'alysl" ha~ o(tutTpd wtlldl (ould translate Into lncrrd<,ed Ct'op pt'otectlOl1. 1\\ thic., pOInt, PXPPI'IIlH'nh undPI' field condItions would havr to OC' calTIPd out tn tl",! tilt' ',1)',1111'> ,1'. possible candlda:e" for Cdrrot (rop protf'ct 10n Thp fot'lllu1.1I10f) 01 tht', l' stralns 1<; also ct'ltlcal for flpld effHelCY It 1'> rl!'«'''\<1l'Y th,lt Ihp Inseet Ingest a lethdl do~e bpfor(' thpy stop fp('dlnq lhp baits and feedln~1 stImulant<, [ould be ddv,HlIdqPOll" d', ,1111.\(1.1111' .. III addltlon, the protec.tlon of thp bacü't'lal IlIlxtllrp Irom '.llnllqhl wOllld bl' essential Slnce these are photoSerlSltlvP On!' fdclor th,lI w(Jllld Ill' lI',Pllll • ta exanllne j:, the effect of th(l ~,trdlll', on ,\lJIIII wr'pVII 11'(lllldlly 01 insects slJrvlvlng the tOXIIl'>. AlttWIHJh dtl ,!ttl'mpt dt Uldt hd'. tJf>j'!I IIIddp, the study failed to Yleld dlly sUltdble re',ult'). Hlf' dect'{!d'>l' III lIll' t'rlle of oviposition of It\',Nts ,;ubJPdpej tn thr' pdtho(jPn wOllid tH' of qrPJt beneflt in the control of thl~ pest. StudYlllg the mode of aetleHI of the bdctf!riullI dqdinst thl~, p(",1 lead', to a bettf'r ulldr>r~, t dnd Hie, 0 r th!' mfl( han 1Sni', 1nvo 1ver! in tox III t Y r h(> Importance of solublllty to tOXIClty should he t!!~ted by ft'pdiWJ ddull weevlls prevlollsly solublliled ItPs. It should aho tH: df'tprrnlllf'd, ll',JnrJ punfieu lep, If thf> r{Jrtot vJepvII rpqulrf", boUt ',pOrf!', dnd (ry',ldl', If) br' effectlvely managed Thl:' would hdve sub',tdntldl HTlpdCt orl t.br> Il',(> of transgenlc Cdrrot" (lmtallllng unly the ,j-f'ndoto/ITI. rhe 66-67 klJd Ilr(J!f'lrl band lS known to be th!: tOYlC mOlf: t of 13. thurTnqlrr}''/', >,utJ'Jp. tenebnon/s and shüuld be testerJ for tO":lClty HI ',traill', A"30 ,!flrJ 1\479. The Bac/llus thunng/ens/s strdln~ hold proml',p a', a uHitrol aqr:nt of the carrot weevll • 100 • APPENDIX • • 101 • e Siological Products Dr. 'LN. Yule ICI Bloloqlcal Product .. Department of Entomology NOlth AIl .. 'II, ,III Faculty of Agriculture Il '1 t If Hl ,ri l l'Ill' t' Macdonald College .'1(11 Il,H1\\t'll 1Il',ld MeGill University ~ ,lit '11\1 l' l ",,, k 21,111 Lakeshote Raad t\11',',I" ,1111),\ l )1\!.IIHl Ste Anne de Bellevue, PO (' ,,1 \, Il 1. 1 l ',)\ ,'1 \ H9X lCO TI'II'pllllIH' (,1111\ IL' \ ,'II,ll 1 dX (,11\,) IL ',1 Oll,1·1 Ref: DJG/File IA3-14-02 Ortobel 25, 19<10 Deal' Dr. Yule, Please finri enclosed sIx Bacillus thurin~~~:: <;traiB'; (Al0, A2 Q Q, A \11, AI,O!) , A410, A429) on Nutrient Agar slants. The<>e "tI,lins havp bppn hoLltpo hele at the ICI Biological Products, NATC, lauoLat(Hy .11ld ale natlll.tl l·,n}.,If", flom Canadian soi1 or grain samplpc;. 111(> <;ix r;tt.till', ...,PIP ,;1\(\...,11 If) 1r.\VP ,H t Ivity against various insect spe<:les in ill',l!cl IJlo.I',' .. IY ',e'l(,('lllll~~. Regarding yOllr letter sent to DL C,11111()11 ddtl'd ()I tolJPl 1flth; ll1rfollllll,ll .. ly, thf' strains which we are sending ltilVf' Ilot bpPI. idf'lIt i f Ipd ,'le. Il. _th~1I illglpl\r;!' tenebrionü,. 1ÎIPse are natuléll i~ol.1I(>', of !J. t. wltirh hilV(' I>P(>II <,ho\oJl\ ln Il,. • active againc,t colcopteran il1',('( l "\)('( \('; hl' bio,\<,',,,y. Ail of tlH' -,11.1111'. ',l'lit have been cho',cll fo thi e, l!rar.le te! l~,t il J hop!' titi', i', Ilot il di~,lpp()llItrnf'lIt to yOll. If ynu leally tH'f..'d ,11\ i lf't\tifH'd Ir'IIf>!J, iOlli', fultlllf' ..... P e,1I1 ,\1«(>';', one for yO\l through anothpl divl~,ion ot ICI; ',() pl!' C As a remltHlcl-, the r1L'l.1I1', (Jf our .1f;reelTlPnt '.Illh IPy"lld to tllf"'" ';II.llI1 , e,\ll tH' found in tire SeCrE.'lY A/,:[('PIn('lIt wll1clr i~, al',o PlI( IU',f'd. If you have any fl\lther qup<,tioll<; reeard:inE" tllP',(> fllltlfres. plp'\',j> Ippl Irr!f' to contact eithet Dr. Roger Bernier or mf'. \Jp look for1Jard to turther di'iCWi·;ioll'i. Sincerely, Lori Moser Microbiologist Encl. • HD-MISC/I090.252 c.e. R.L. Bernier • ICI Biological Pr{)ducls ~JOllll Alli' 'II: III 1(',11111'" JI ( ""tl" 21(J1 H,I:l,YI Il HUdcJ Sllf'llrl,l' 1',1'" r1>11' r,!' ,,l''n,} OI1!.l',() Ms. Fabianne Saade Célll.ld 1 l ',f\:'l l Dept. of Entomology 1«lcpll(I[I« rcl 1r,l 1)23 ;71 GO MacDonald Callege f d Y (1 Fl) ECJ 0044 21111 Lak~5hare Rd. Ste. Anne de Bellevue, P.Q. H9X lCa December 13, 1990 Dear Ms. Saade, Enclased in this package you will find 2 nutrient agar slants inoculated with our control straln of B.t. tenebrionlS. Before sendinq thp rtrain ta you l have checked its purlty, Gram stain and .1bllity t0 produc0 cryr;tals. The straln i5 charactcx:-ist • ically a Gr"m pOC;ltlV!! rad that produces a large cpntral spore and small C[y:.têll~) upon lyS1S. The lsolated colony morphology ir; crf'amy white wlth Itreqular edges. In general, aIl B.t. strain~ q[nw bcst at 30°C êlnd wlli enter lysis stage on day 3 or '1 • Good luck ln your research and do not hesitate to calI If you have any questions . •