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The implications of cabbage looper (Trichoplusia ni) : nuclear polyhedrosis virus coevolution for biological… Milks, Maynard Lionel 1996

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THE IMPLICATIONS OF CABBAGE LOOPER (Tr ichop lus ia n i ) - NUCLEAR POLYHEDROSIS VIRUS COEVOLUTION FOR BIOLOGICAL CONTROL by MAYNARD LIONEL MILKS B. S c . , The Un ivers i ty of Ottawa, 1986 M. S c . , The Un ivers i ty of Ottawa, 1988 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES (Department of Zoology) We accept t h i s thes is as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August 1996 (c)Maynard L ione l M i l k s , 1996 In presenting this, thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia Vancouver, Canada Date ND\f£M&£fL lC, tftJL DE-6 (2/88) i i ABSTRACT Many features make nuclear polyhedrosis v i ruses (NPVs) an a t t r a c t i v e a l te rna t ive to chemicals as i n s e c t i c i d e s . However, perhaps the s i n g l e most appealing feature of NPVs i s the p o s s i b i l i t y that one app l i ca t ion of v i r u s could lead to long-term cont ro l of the pes t . This i s because fo l lowing an i n i t i a l i n o c u l a t i o n , v i r a l p a r t i c l e s w i l l be re leased in the environment at each insect generat ion, assur ing a continuous supply of inoculum. Sustained exposure to NPVs i s l i k e l y to s e l e c t for res is tance . However, i n contrast to chemicals, t h i s might not pose such a problem as NPVs can a lso evolve and become more v i r u l e n t . Assessing the coevolut ionary t ra jec to ry of i n s e c t -NPV assoc ia t ion i s thus c r i t i c a l i n determining the po ten t i a l of these v i ruses as long-term contro l agents. In my t h e s i s , I examined the coevolut ion of cabbage loopers (T r i chop lus ia n i Hubner) and i t s associated singly-embedded nuclear polyhedrosis v i r u s (TnSNPV). I chose T. n i because i t i s a genera l i s t herbivore of great economic importance and because i t has a short generat ion time thus making i t a good candidate for studies of evo lu t ion . i i i Resistance to TnSNPV w i l l evolve i f s u s c e p t i b i l i t y var ies among ind iv idua ls and i f i t i s g e n e t i c a l l y determined. In Chapters 1 and 2, I showed that the s u s c e p t i b i l i t y of c a t e r p i l l a r s to TnSNPV var ied among and wi th in populat ions of T. n i and that i t was a her i t ab le t r a i t . In Chapter 3, I conducted an experiment on cabbage looper-TnSNPV coevolut ion . In a l l three r e p l i c a t e s , T. n i evolved res is tance to TnSNPV, but in none of the r e p l i c a t e s d id the v i r u s become s i g n i f i c a n t l y more v i r u l e n t . Resistance to pathogens i s f requent ly be l ieved to be c o s t l y . In Chapter 4, I observed that T. n i from cont ro l and se lected l i n e s reared in the absence of v i r u s had s i m i l a r pupal weight, developmental t ime, egg product ion and egg h a t c h a b i l i t y . Chapter 5 was a departure from the theme of coevolut ion . The purpose of t h i s chapter was to determine i f the subletha l e f f e c t s of TnSNPV were a funct ion of dose and the age at which c a t e r p i l l a r s contract the d isease . I observed that the pupal weight, developmental time and egg production of i nd iv idua ls in fected at 3rd ins ta r were impaired r e l a t i v e to c o n t r o l s . In a d d i t i o n , I found these subletha l e f f ec ts were independent of TnSNPV dose. F i n a l l y , I iv observed that the v i r u s d i d not have any d e b i l i t a t i n g e f f e c t s on survivors when T. n i were treated at 4th or 5th i n s t a r . These findings suggest that T. n i can evolve resistance to TnSNPV and that resistance i s l i k e l y to be stable and not to decline when the virus i s removed. F i n a l l y , a pplication of the virus against early T. n l instars may res u l t i n more e f f i c i e n t control because young c a t e r p i l l a r s are more susceptible to TnSNPV and because survivors may have impaired reproductive success. V TABLE OF CONTENTS Abstract i Table of Contents v List of Tables v i List of Figures V H Acknowledgments INTRODUCTION x 1 Chapter One The Comparative Biology and S u s c e p t i b i l i t y 14 of Cabbage Looper (T r ichop lus ia n i ) Populat ions to the Singly-Embedded Nuclear Polyhedrosis V i r u s of T. n i Chapter Two V a r i a t i o n in S u s c e p t i b i l i t y 50 to the Singly-Embedded Nuclear Polyhedrosis V i rus of T r i c h o p l u s i a n i Within a Populat ion of Cabbage Loopers Chapter Three Keeping Up with the Red Queen: 65 Cabbage Looper - Nuclear Polyhedrosis V i rus Arms-Race Chapter Four Is Resistance to the Singly-Embedded 121 Nuclear Polyhedrosis V i rus of T r i c h o p l u s i a n i Cost ly? Chapter Five The E f f e c t of Larval Age on the Sublethal 144 E f f e c t s of the Singly-Embedded Nuclear Polyhedrosis V i rus of T r i c h o p l u s i a n i on Cabbage Loopers CONCLUSIONS 176 Bibliography 186 Appendix One The E f f e c t of Ingestion Time of Treated 225 Plugs on the S u s c e p t i b i l i t y of Cabbage Loopers to the Singly-Embedded Nuclear Polyhedrosis V i rus of T r i c h o p l u s i a n i v i L i s t of Tables 1.1 H is tory of cabbage looper popula t ions . 34 1.2 Diet formulat ion for cabbage looper la rvae . 35 1.3 Comparison of slopes and in tercepts of dosage- 36 mor ta l i ty curves of the 12 cabbage looper populat ions. 1.4 Three-way ANOVA for the durat ion of l a r v a l and 37 pupal stages and pupal weight of i n d i v i d u a l s surv iv ing treatment with TnSNPV. 1.5 TnSNPV dose (number of OBs) which resu l ted in 39 s i g n i f i c a n t v a r i a t i o n in pupal weight by the Tukey mul t ip le range t e s t . 1.6 Pearson's r ( 1 - t a i l p r o b a b i l i t y ) r e l a t i n g mean 40 l i f e - h i s t o r y t r a i t s of males and females to the LD50 of t h e i r l i n e (N=10). 2.1 Number of f a m i l i e s ( f ) , mean number of larvae per 64 family (n), mean percent mor ta l i ty per family (P) , estimate of h e r i t a b i l i t y ( h 1 ) , and chi -square s t a t i s t i c for genet ic var iance . 3.1 LD50 [95% c . i . ] ca lcu la ted by SAS for contro l and 101 se lected l i n e s 1 and 2 at generations 7-11 and 20. 5.1 Percent s u r v i v a l , LD50 and slope and intercept of 164 dosage-mortal i ty curves in r e l a t i o n to the age at which cabbage looper larvae were in fec ted . 5.2 Four-way ANOVA of the pupal weight of i n d i v i d u a l s 165 surv iv ing to adulthood with TnSNPV dose (DOSE), l a r v a l age at i n f e c t i o n (AGE), sex (SEX) and t r i a l (TRIAL) as p red ic to r v a r i a b l e s . 5.3 Four-way ANOVA of the time to adult emergence 166 with TnSNPV dose (DOSE), l a r v a l age at i n f e c t i o n (AGE), sex (SEX) and t r i a l (TRIAL) as pred ic tor v a r i a b l e s . 5.4 Three-way ANOVA of the number of eggs l a i d per 167 female with TnSNPV dose (DOSE), l a r v a l age at i n f e c t i o n (AGE) and t r i a l (TRIAL) as p red ic to r v a r i a b l e s . A . l Modal plug (N) consumption time (hr) of surv iv ing 229 males (M) and females (F) and larvae succumbing (X) to TnSNPV. v i i L i s t of Figures 1.1 LD50 and 95% confidence limits of each cabbage 41 looper population 1.2 Mean (+1 SE) duration of the larval stage (days) of 43 surviving females (•) and males (•) averaged across a l l TnSNPV doses for each cabbage looper population. 1.3 Mean (+1 SE) duration of the pupal stage (days) of 45 surviving females (•) and males (•) averaged across a l l TnSNPV doses for each cabbage looper population. 1.4 Mean {±1 SE) pupal weight (mg) of surviving females 47 (•) and males (•) in relation to TnSNPV dose for each cabbage looper population. 3.1 Flowchart summary of selection experiments and 102 assays used to detect changes in the susceptibility of cabbage loopers to TnSNPV and in the virulence of TnSNPV. 3.2 Percent survival of lines during selection. (A) Mean 104 (+1 SE) percent survival of control (A) and selected (•) lines 1 and 2. There was no selection between generations 9 and 14. (B) Percent survival of control (•) and selected (•) line 3. 3.3 (A) Mean (+1 SE) percent survival of larvae from 106 control (•) and selected (•) lines 1 and 2 inoculated with 11000 OBs of TnSNPVl-GO for generations 2 to 6. (B) Mean (+1 SE) LD50 of control (A) and selected (•) lines 1 and 2 treated with TnSNPVl-GO. 3.4 (A) Survival of larvae from control (A) and selected 108 (•) line 3 treated with 7500 OBs of TnSNPV3-G0 for generations 1-15. (B) LD50 and 95% confidence intervals of control (A) and selected (•) line 3 infected with TnSNPV3-G0. 3.5 Mean (+1 SE) percent survival of larvae from control 110 line 1 treated with 1000 (A) or 2000 OBs (•) of TnSNPVl or TnSNPV2 from generations 0, 1, 2, 5, 7, 13, 16 or 19. v i i i 3.6 Percent s u r v i v a l of larvae from cont ro l l i n e 3 112 in fected with 14000 OBs of TnSNPV3 from (A) generations 0, 1, 2, 4, 5, 6, 10 or 15 (Assay 1) and; (B) generations 0, 5, 9, 11, 13 or 14. (C) Assays 1 and 2 combined. 3.7 R e s t r i c t i o n endonuclease p r o f i l e s of the DNA of the 114 5 TnSNPV i s o l a t e s : (1) o r i g i n a l v i r u s for l i n e s 1 and 2, (2) se lected v i rus from l i n e 1 generation 19, (3) se lec ted v i r u s from l i n e 2 generation 19, (4) o r i g i n a l v i rus for l i n e 3, and (5) se lected v i r u s from l i n e 3 generat ion 15 digested with e i ther (A) BamHI, (B) EcoRI, (C) H i n d l l l , (D) Ps t I , (E) S a i l and (F) Ss t I . 4.1 Mean (±1 SE) pupal weight (mg) of (A) females and 132 (B) males from cont ro l and se lec ted l i n e s 1 and 2. 4.2 Mean (+1 SE) developmental time (days) of (A) 134 females and (B) males from contro l and se lec ted l i n e s 1 and 2. 4.3 Mean (+1 SE) pupal weight (mg) of (A) females and 136 (B) males from cont ro l (A) and se lec ted (•) l i n e s 3 at generations 14 and 16. 4.4 Mean (+1 SE) developmental time (days) of (A) 138 female and (B) males from cont ro l (•) and se lected (•) l i n e s 3 at generations 14 and 16. 4.5 Mean (±1 SE) egg production of females from cont ro l 140 (A) and se lected (•) l i n e s 3 at generations 13-17. 4.6 Mean (+1 SE) percent hatch of eggs l a i d by females 142 from cont ro l (•) and se lec ted (•) l i n e s 3 at generations 16 and 17. 5.1 Adjusted mean (+1 SE) pupal weight (mg) of (A) 168 females and (B) males surv iv ing to adulthood i n r e l a t i o n to TnSNPV treatment and the age in days at which larvae were inocu la ted . 5.2 Adjusted mean ( i l SE) developmental time (days) of 170 (A) males and (B) females surv iv ing to adulthood i n r e l a t i o n to TnSNPV treatment and the age in days at which larvae were inocula ted . ix 5.3 Mean (+1 SE) egg product ion per female in r e l a t i o n 172 to TnSNPV treatment and the age i n days at which larvae were inocula ted . 5.4 Mean (+1 SE) percent egg hatch i n r e l a t i o n to TnSNPV 174 treatment and the age i n days at which larvae were inocula ted . X Acknowledgments I would l i k e to thank my superv isor , Judy Myers, fo r her tremendous pat ience, for near ly g iv ing me a blank check for buying laboratory suppl ies (8.31 x 1C? p l a s t i c cups and 2.49 metr ic tons of agar) and for her e d i t i n g which grea t ly improved the c l a r i t y of my t h e s i s . I am gra te fu l to my research committee, Drs. Mart in Adamson, Dolph Sch lu ter , Geoff Scudder and David Theilemann for t h e i r assistance throughout my Ph. D. s tudies and for commenting on ear ly vers ions of my t h e s i s . S p e c i f i c a l l y , I would l i k e to thank Dr. Adamson for a l lowing me to store ingredients and to prepare the growth media fo r cabbage looper larvae in h i s laboratory . Dr. Scudder a lso provided space in h i s laboratory to store cabbage looper eggs and some d ie tary ingred ients . I am gra te fu l to Dr. Schluter for d i s c u s s i o n and fo r ass is tance with s t a t i s t i c s . I would l i k e to thank Dr. Theilmann for a l lowing me to use h i s equipment and showing me how to i s o l a t e v i r u s and d igest TnSNPV DNA. Sandy Stewart, in p a r t i c u l a r , but a lso Cindy Shipman and Ian Forsythe ( a l l Theilmann lab) a lso helped me considerably with the d i g e s t s . Many people helped me rear cabbage loopers ( in order of x i appearance): Terry Gambrel, Jenni fer Boyer, Constance Himmack, Steve Connor, S h i r l e y Tsang and Je f fe rey Florando. Three people deserve a s p e c i a l accolade: Thuy Nguyen and Igor Burnstyn worked for me 3 1/2 and 5 years , r e s p e c t i v e l y . Both of them were very dedicated, met iculous, r e l i a b l e and trustworthy. They were extremely e f f i c i e n t and I cannot r e c a l l e i ther of them making a mistake. Thei r conversat ion was always d e l i g h t f u l and in te res t ing and t h e i r sense of humor made time go by much f a s t e r . One would be hard pressed to f i n d bet ter a s s i s t a n t s . I wish them both the best of luck with t h e i r graduate s t u d i e s . Last but c e r t a i n l y not l e a s t , I would l i k e to thank my wi fe , Miche l le Lep t ich . No words w i l l s u f f i c e to express my grat i tude and t h i s thes is would not have been poss ib le without her . She helped me countless hours i n the l ab , found and photocopied a r t i c l e s , entered data, proofread over and over , prepared many meals for me and even de l ivered some to the lab . She was always very support ive even when i t meant that I had to go back to the lab at n igh t , during the wee hours or on weekends. Her understanding and encouragement made th ings much eas ie r for me. She should get an honorary degree jus t fo r what she endured and went through. I am g r a t e f u l to Drs . Br ian F e d e r i c i (UC I rv ine , x i i I rv ine , C a l i f o r n i a ) , Pa t r ick Hughes (Cornel l U n i v e r s i t y , I thaca, New York) , Car lo Ignoffo (USDA-ARS, Columbia, M i s s o u r i ) , Robert Jaques (Agr icu l ture Canada, Harrow, Ontar io ) , Wi l l iam Kaupp (Forestry Canada, Sault Ste-Mar ie , Ontario) and Andrew Keddie (Univers i ty of A l b e r t a , Edmonton A l ) , Mr. Francois Fournier (Agr icu l ture Canada, S t - J e a n - s u r -l e R i c h e l i e u , Quebec), Mr. Daniel Hogue (Sandoz Crop Pro tec t ion , Wasco, C a l i f o r n i a ) and Ms. L i l i a n Moog (USDA-ARS, Phoenix, Arizona) fo r sending me cabbage looper eggs or pupae. I am gra te fu l to the owners of the Hazelmere and Langley greenhouses (Langley, B. C.) fo r a l lowing me to go and c o l l e c t cabbage looper larvae . Mr. Mark Sweeney (B.C. M in is t ry of Agr icu l ture ) a lso arranged access to a cabbage patch in Abbotsford where I c o l l e c t e d c a t e r p i l l a r s . Mr. Dan Laing (Agr icu l ture Canada, Harrow, Ontario) and Ms. Paula Peters (USDA-ARS, Columbia, Missour i ) gave me many good h in ts on how to rear cabbage loopers in the laboratory . Dr. Jaques provided the TnSNPV samples I used to conduct my experiments. I would l i k e to thank Richard Kinkead, Joe l Sawada, Troy S i t l a n d , Simon E l l i s , Richard C o l l i n , John Adamick and Duncan McFarlane for dragging me away from the lab to play hockey or go l f and without whose ass is tance t h i s thes is would have been f i n i s h e d much e a r l i e r . x i i i This study was supported by a Natural Sciences and Engineering Research Counci l of Canada postgraduate scholarsh ip to MLM and an operat ing grant to Judy Myers. INTRODUCTION The e f f i c i e n t cont ro l of phytophagous insects i s c r i t i c a l to successfu l agr icu l tu re and s i l v i c u l t u r e (Simpson and Conner-Ogarzly 1986). Since t h e i r advent, chemical i n s e c t i c i d e s have been the predominant means for con t ro l of herbivorous insects (Wilcox et a l . 1986). Although cred i ted with increas ing crop y i e l d s , numerous problems have a r isen with the use of chemicals (e .g . Carson 1962): (1) appearance of res is tance in target species (more than 500 species of insects are now r e s i s t a n t to at l eas t one chemical i n s e c t i c i d e ; Roush and Tabashnik 1990), (2) extensive s i d e -e f f e c t s (genetic damage and/or death) in non-target invertebrate s p e c i e s , (3) accumulation of t o x i c residues i n the environment and, (4) substant ia l monetary costs (Simpson and Conner-Ogarzly 1986). These problems prompted a thorough review of pest management s t ra teg ies and led to the development of bet ter a g r i c u l t u r a l p r a c t i c e s , integrated pest management programs and to more ser ious considerat ions of b i o l o g i c a l agents ( e .g . predators , p a r a s i t o i d s , b a c t e r i a , v i r u s e s , e t c . ) as a l te rna t ives to chemicals for c o n t r o l l i n g insec ts (WHO 1973, Bastra 1982). Nuclear polyhedrosis v i ruses (NPVs; family Baculovir idae) are pathogens that mainly i n f e c t lepidopteran 2 l a rvae , and have received considerable a t tent ion as p o t e n t i a l b i o l o g i c a l i n s e c t i c i d e s . NPVs cons is t of a double-stranded, c i r c u l a r , supercoi led DNA genome of 90 to 200 k i lobases enclosed in a proteinaceous capsid ( e .g . Volkman and Keddie 1990). The DNA-capsid complex i s known as a nucleocapsid and one (singly-embedded) to severa l (multi-embedded) nucleocapsid(s) i s / a r e surrounded by a l i p o p r o t e i n membrane to form a v i r i o n . The v i r i o n s are enclosed in a p a r a c r y s t a l l i n e g lycoprote in matrix, the whole being known as an i n c l u s i o n or occ lus ion body. The other member of the Baculov i r idae family i s the granulos is v i r u s (GV). GVs a lso cons is t of a c i r c u l a r , double-stranded DNA genome but only have 1 nucleocapsid per v i r i o n and 1 v i r i o n per occ lus ion body (e .g . Evans and Entwist le 1987). The l i f e cyc le of NPVs usua l ly begins with insects ingest ing food contaminated with occ lus ion bodies ( e .g . Mazzone 1985, Evans and Entwist le 1987). There are severa l other poss ib le routes of entry for the v i r u s in the host . F i r s t , the v i r u s may be passed from mothers to o f f s p r i n g wi th in the egg ( t ransovar io l t ransmiss ion; e . g . Shapiro and Roberstson 1987). Second, larvae may ingest the v i r u s at hatch when they chew through the eggshel l (transovum t ransmiss ion) . V i rus may be deposited on the eggs at o v i p o s i t i o n by moths that are in fected or ex te rna l ly 3 contaminated (see Murray and E lk in ton 1989). A l t e r n a t i v e l y , v i r u s that i s in the environment may be leached by the r a i n or blown by the wind onto eggs (Murray and E lk in ton 1989). T h i r d , p a r a s i t o i d females may vector the disease upon p a r a s i t i z i n g in fec ted larvae ( e .g . Levin et a l . 1979, Young and Yearian 1990). The fo l lowing i s a summary of the i n v ivo pathway of i n f e c t i o n of the archetype NPV, Autographa c a l i f o r n i c a NPV. Once in the midgut, the occ lus ion body matrix i s d i s s o l v e d , r e s u l t i n g in the re lease of v i r i o n s ( e . g . Granados and Lawler 1981, Mazzone 1985, Evans and Entwist le 1987, B l i s s a r d and Rohrmann 1990). This i s an e s s e n t i a l step s ince the v i r i o n s and not the occ lus ion bodies per se are the in fec t ious uni t of NPVs (e .g . Mazzone 1985, Evans and Entwist le 1987, B l i s s a r d and Rohrmann 1990). The l ibe ra ted v i r i o n s , c a l l e d polyhedra-der ived v i r i o n s (PDV), are very suscept ib le to i n a c t i v a t i o n by the d iges t ive proteases of the insect (Elam et a l . 1990), and must r a p i d l y pass through the p e r i t r o p h i c membrane (PM) to reach the midgut sur face . The PM i s a s i e v e -l i k e s t ructure that surrounds the food as i t enters the midgut ( e .g . Wigglesworth 1972). It i s composed p r i n c i p a l l y of glycosaminoglycans, prote ins and hyaluronic ac id embedded i n c h i t i n f i b r i l s and protects the midgut epi thel ium from abrasion by food p a r t i c l e s . The PM may a lso provide a 4 mechanical b a r r i e r to invading microorganisms (Adang and Spence 1981). It has been suggested that NPVs possess a pro te in that can d isrupt the PM to allow free passage of the v i r i o n s to the surface of the midgut (Derksen and Granados 1988). PDV enter midgut columnar e p i t h e l i a l c e l l s by fus ion with the plasma membrane (Granados and Lawler 1981). PDV may even i n f e c t the regenerat ive c e l l s of the midgut as primary targets (Keddie et a l . 1989). It i s not known how PDV reach the regenerative c e l l s s ince they are not exposed to the midgut lumen (Volkman and Keddie 1990). The v i r i o n s produced in the midgut, c a l l e d e x t r a - c e l l u l a r v i r i o n s (EV), contain only one nucleocapsid and are not occluded. Although g e n e t i c a l l y i d e n t i c a l , PDV and EV d i f f e r in morphology, time and place of maturation, s t ruc tu re , a n t i g e n i c i t y and i n f e c t i v i t y (Volkman et a l . 1976). EV i n i t i a l l y i n f e c t the t racheal system and appear to use i t as conduits to enter the c e l l s of other t i ssues and to spread the disease systemica l ly (Engelhard et a l . 1994). The v i r i o n s that are produced in the l a te stage of the disease are occluded ( e . g . B l i s s a r d and Rohrmann 1990). The sequence of events t r i g g e r i n g the occ lus ion of v i r i o n s i s not f u l l y understood. U l t imate ly , in fec ted hosts d ie and l y s e , re leas ing occ lus ion bodies in the environment that can l a t e r serve as inoculum for other i n s e c t s . The occ lus ion body matrix protects the v i r i o n s from i n a c t i v a t i o n by u l t r a v i o l e t l i g h t ( e .g . Entwist le and Evans 5 1985) and allows them to p e r s i s t i n the environment fo r up to 40 years (Thompson et a l . 1981 but see Jaques 1977a, Oogard et a l . 1988). NPVs are the only known v i ruses to use two phenotypes to complete t h e i r l i f e cyc le ( e .g . Volkman and Keddie 1990) . Many features make NPVs an a t t r a c t i v e a l t e rna t i ve to chemicals, p a r t i c u l a r l y against pest lepidopterans and several NPVs are cur rent ly reg is tered with the United States Environmental Protect ion Agency as b i o l o g i c a l i n s e c t i c i d e s (Podgwaite 1985). F i r s t , NPVs are n a t u r a l l y o ccu r r in g , h igh ly v i r u l e n t pathogens (e .g . Pogwaite 1985; Evans and Entwist le 1987). NPV ep izoot ics have f requent ly been associated with c o l l a p s i n g populat ions of insects ( e .g . Steinhaus 1949, B i r d and Burk 1961, Campbell 1963a, Doane 1969, 1970, 1976, Burgess and Hussey 1971, S t a i r s 1972, Leonard 1974, Anderson and May 1980, Woods and E lk in ton 1987, E lk in ton and Liebhold 1990, Myers 1993 see a lso Marshal l 1970, Fuxa and Geaghan 1983, Zelazny et a l . 1992). Second, NPVs are pathogens s p e c i f i c to arthropods with 70% of known NPVs having been i s o l a t e d from lepidopteran larvae ( e . g . Evans and Entwist le 1987). The majori ty of the remaining NPVs have been recovered from sawf l ies (Hymenoptera; Evans and Entwis t le 1987). Hence, NPV i n s e c t i c i d e s may be l ess l i k e l y to harm b e n e f i c i a l insec ts ( p a r a s i t o i d s , predators and p o l l i n a t o r s ) , plants or vertebrates than many chemical i n s e c t i c i d e s (Dol ler 1985, Evans and Entwist le 1987). T h i r d , perhaps the s i n g l e most promising feature of NPVs as an a l t e rna t i ve to chemicals i s the p o s s i b i l i t y that a s ing le a p p l i c a t i o n of v i r u s could r e s u l t i n long-term suppression of the pest (Podgwaite 1985). This i s because fo l lowing an i n i t i a l i n o c u l a t i o n , v i r a l p a r t i c l e s w i l l be re leased in the environment at each insect generat ion, thus assur ing a continuous supply of inoculum. The success of NPVs as long-term pest cont ro l agents w i l l depend on the coevolut ionary t ra jec to ry of the host insect and the v i r u s . E h r l i c h and Raven (1964) introduced the term coevolut ion to descr ibe how plants and herbivores may a f f e c t each o t h e r ' s evo lu t ion . Janzen (1980) def ined coevolut ion as an evolut ionary response in populat ion A r e s u l t i n g from i t s in te rac t ion with populat ion B, fol lowed by a genet ic change in B r e s u l t i n g from the evolut ionary response in A. Long-term exposure of insects to NPVs i s l i k e l y to s e l e c t for res is tan t i n d i v i d u a l s . Although there have only been a few reports of res is tance to NPVs (Martignoni 1957, Fuxa et a l . 1988 but see Ignoffo and A l l e n 1972, Whitlock 1977, Kaomini and Roush 1988) and other baculoviruses (Rivers 1959, Sidor 1959, David and Gardiner 1965, Br iese and Mende 7 1981 see a lso Zelazny et a l . 1989), the po ten t ia l for such res is tance to evolve does e x i s t (Briese 1986). Resistance to NPVs w i l l evolve i f s u s c e p t i b i l i t y var ies among i n d i v i d u a l s and i f i t i s g e n e t i c a l l y con t ro l l ed (e .g . Endler 1986). Resistance to pathogens may be c o s t l y and may involve the d i v e r s i o n of energy from growth and/or reproduct ion to combat the pathogen (see Dawkins and Krebs 1979, Futuyma 1983, Thompson 1986, Simms and Rausher 1987, Simms and F r i t z 1990, Toft and Karter 1990). The cost of res is tance w i l l p lay an important ro le in the evolut ion of r e s i s t a n c e . I f res is tance i s c o s t l y , and r e s i s t a n t i n d i v i d u a l s have a lower reproductive success than uninfected suscept ib le ones, then the evolut ion of res is tance may be constrained (Thompson 1986, Tof t and Karter 1990). Lenski and Levin (1985) se lec ted Escher ich ia c o l i fo r res is tance to bacteriophage T4. As the proport ion of E .^ c o l i genotypes r e s i s t a n t to T4 increased fo l lowing s e l e c t i o n , resources were depleted and a small populat ion of suscept ib le in d iv id u a ls remained. Lenski and Levin (1985) suggested that some suscept ib les pers is ted because they could u t i l i z e l i m i t i n g resources more e f f e c t i v e l y than r e s i s t a n t E .^ c o l i i . e . suscept ib le bac te r i a were super ior competitors at low resources. In a re la ted matter, Berenbaum et a l . (1986) showed that furanocoumarins protected the wi ld parsnip (Pastinaca sat iva) from herbivory 8 by the parsnip webworm (Depressaria pas t inace l l a ) and was p o s i t i v e l y cor re la ted with seed set when the herbivore was present . However, they a lso observed that P_;_ s a t i v a p lants with high l e v e l s of furanocoumarins produced fewer seeds when webworms were absent, and concluded that the negative genet ic c o r r e l a t i o n among f i t n e s s and res is tance characters ( i . e . the cost of res istance) could l i m i t the evolut ionary response of the host . The evolut ionary t ra jec to ry of the v i r u s in insect-NPV coevolut ion i s even more d i f f i c u l t to p r e d i c t . The "Conventional Wisdom" (Anderson and May 1982) i s that paras i tes should evolve to do l i t t l e or no harm to t h e i r host and that commensalism i s the only s table coevolut ionary outcome for hos t -paras i te a s s o c i a t i o n s . This i s because death of the host i s associated with death of the p a r a s i t e . Obviously , such an outcome would severely r e s t r i c t the e f f i c a c y of NPVs as long-term pest cont ro l agents. However, recent hos t -paras i te models suggest that severa l coevolut ionary outcomes are poss ib le for v i r u s e s , among which i s increased v i ru lence ( e . g . Levin and Pimental 1981, Anderson and May 1982, Bremermann and P icker ing 1983, May and Anderson 1983). If t h i s was the case, the evolut ion of res is tance in c a t e r p i l l a r s would not a f fec t the e f f i c a c y of NPVs as long-term pest cont ro l agents because the v i r u s may 9 be able to counteract the res is tance of the host by becoming more v i r u l e n t . Determining the coevolut ionary t ra jec to ry of insect-NPV assoc ia t ions i s thus c r i t i c a l i n evaluat ing the po ten t ia l of these v i ruses as long-term pest cont ro l agents. The o v e r a l l goal of my t h e s i s was to determine the coevolut ionary outcome of a lepidoptera-NPV a s s o c i a t i o n . W i l l c a t e r p i l l a r s evolve res is tance when continuously exposed to NPVs? And, how w i l l t h i s feedback on the v i rus? To answer these questions and reach my g o a l , I conducted a s e r i e s of laboratory experiments examining d i f f e r e n t facets of lepidoptera-NPV coevolut ion . I se lected cabbage loopers (T r ichop lus ia n i Hubner) as my lepidopteran model for severa l reasons: f i r s t , T. n i can be cul tured continuously without diapausing. Second, laboratory rear ing techniques inc lud ing a semi-synthet ic d i e t for la rvae , are wel l es tab l ished (Ignoffo 1963). T h i r d , the generation time of cabbage loopers at 26°C i s about 22-24 days. Thus, i t i s poss ib le to conduct a f a i r l y long coevolut ion experiment (15-20 generations) in a reasonable length of time (1.5-2 y e a r s ) . Fourth, T . n i i s suscept ib le to an NPV, the s i n g l y embedded nuclear polyhedrosis v i r u s of T. n i (TnSNPV), which shows a great deal of po ten t ia l as a b i o i n s e c t i c i d e against cabbage loopers (Jaques 1972, 1977b, Entwist le 1983). F i f t h , T. n i i s one of the most ser ious d e f o l i a t o r s of cole crops and i s a pest of 10 economic importance (Lindgren and Greene 1984). I d iv ided my thes is into 5 chapters. Chapter One i s e n t i t l e d "The Comparative Bio logy and S u s c e p t i b i l i t y of Cabbage Looper (T r ichop lus ia n i ) Populat ions to the S i n g l y -Embedded Nuclear Polyhedrosis V i rus of T . n i " . The purpose of Chapter 1 was three f o l d : (1) to determine whether the s u s c e p t i b i l i t y to TnSNPV var ied among 12 populat ions of cabbage loopers;(2) to determine whether the s u s c e p t i b i l i t y of a populat ion to TnSNPV (LD50) was cor re la ted with the f i t n e s s of i n d i v i d u a l s in v i r u s - f r e e environments; (3) to determine i f i n d i v i d u a l s that survived the disease were as f i t as c o n t r o l s . Chapter Two i s c a l l e d " V a r i a t i o n i n S u s c e p t i b i l i t y to the Singly-Embedded Nuclear Polyhedrosis V i rus of T r i c h o p l u s i a n i Within a Populat ion of Cabbage Loopers". The purpose of t h i s chapter was to determine i f the s u s c e p t i b i l i t y of T . n i to TnSNPV var ied wi th in a populat ion of cabbage loopers . To reach t h i s g o a l , 1 examined i f s u s c e p t i b i l i t y was h e r i t a b l e . I i n d i v i d u a l l y mated p a i r s of adu l ts , in fec ted t h e i r progeny with TnSNPV and used a quant i ta t ive genet ic model for a threshold t r a i t with a f u l l s i b l i n g design to ca lcu la te the h e r i t a b i l i t y of s u s c e p t i b i l i t y to the v i r u s . In the f i r s t experiment, I only 11 used adul ts from the Jaques populat ion (one of the 12 populat ions studied in Chapter 1) . Th is populat ion had been cul tured in the laboratory for almost 40 years and may show l i t t l e v a r i a t i o n for s u s c e p t i b i l i t y . In the second and t h i r d experiments, I used adul ts from a populat ion that had recent ly been estab l ished by mixing i n d i v i d u a l s from the 12 cabbage looper populat ions studied in Chapter 1. Th is composite populat ion may show more v a r i a t i o n among in d iv idua ls for s u s c e p t i b i l i t y to TnSNPV and w i l l provide an i n t e r e s t i n g comparison to the Jaques popula t ion . Chapter Three i s e n t i t l e d "Keeping up with the Red Queen: Cabbage Looper - Nuclear Polyhedrosis V i rus Arms-Race". This chapter i s the crux of my t h e s i s . Here, I present the r e s u l t s of two experiments on the coevolut ion of cabbage loopers and TnSNPV. In the f i r s t experiment, I es tab l ished 4 l i n e s (2 contro l l i n e s and 2 se lected l ines ) by taking c a t e r p i l l a r s from 5 of the populat ions I studied i n Chapter 1. I t reated the larvae from the se lected l i n e s with TnSNPV and mass-mated the s urv iv ors . I i s o l a t e d the v i r u s from larvae succumbing to the v i r u s from each l i n e and used t h i s "selected" TnSNPV to inoculate the progeny of the surv ivors of that l i n e . The contro l l i n e s were t reated in the same way except that they were mock-infected with d i s t i l l e d water. I monitored the evolut ion of cabbage looper res is tance 12 to TnSNPV by inocu la t ing cont ro l and se lec ted larvae with the o r i g i n a l v i r u s . I monitored the evolut ion of the v i ru lence of TnSNPV by comparing the s u r v i v a l of cont ro l larvae t reated with the o r i g i n a l or se lected TnSNPV. The second experiment was conducted analogously except that I only had one cont ro l and one se lected l i n e . Moreover, both l i n e s were o r i g i n a l l y es tab l ished by taking i n d i v i d u a l s from a l l 12 populat ions studied in Chapter 1. Chapter Four i s c a l l e d "Is Resistance to the S i n g l y -Embedded Nuclear Polyhedrosis V i rus of T . n i Cost ly? In t h i s short chapter, I compare the developmental t ime, pupal weight and reproductive success of contro l and se lec ted cabbage loopers reared in the absence of TnSNPV. If res is tance to the v i r u s i s reproduct ive ly c o s t l y , I p red ic t that i n d i v i d u a l s from se lec ted l i n e s w i l l have a lower f i t n e s s than cont ro ls when reared i n TnSNPV-free environments. Chapter F ive i s e n t i t l e d "The E f f e c t of Larva l Age on the Sublethal E f f e c t s of the Singly-Embedded Nuclear Polyhedrosis V i rus of T r i c h o p l u s i a n i on Cabbage Loopers". This chapter departs from the theme of coevolut ion . In Chapter 1, I examined the e f f e c t of the v i r u s on developmental time and pupal weight of c a t e r p i l l a r s t reated at 5th i n s t a r . In t h i s chapter , I in fec ted larvae at 3rd , 4th 13 and repeated the experiment with 5th ins ta rs to determine i f the sublethal e f f e c t s of the v i r u s may be a funct ion of the age at which the c a t e r p i l l a r s contract the d isease . I synthesize my f ind ings in the "Conclusions" sec t ion and d iscuss t h e i r impl ica t ions for the use of NPVs as long-term pest contro l agents. There i s a lso one appendix to my t h e s i s . Appendix One i s c a l l e d "The Ingestion Time of V i r u s Does Not A f f e c t the S u s c e p t i b i l i t y of Cabbage Loopers to the Singly-Embedded Nuclear Polyhedrosis V i rus of T r i c h o p l u s i a n i " . Throughout my study, I in fec ted larvae by feeding them a small plug of d i e t contaminated with v i r u s . This technique has been c r i t i c i z e d because larvae that eat the piece of d i e t qu ick ly may have a lower p r o b a b i l i t y of s u r v i v a l than larvae that consume the d ie t slowly (Sai t et a l . 1994a). Hence, the technique per se could a f f e c t the s u r v i v a l of cabbage loopers inoculated with v i r u s . To examine the e f f e c t of ingest ion time on s u r v i v a l , I t reated larvae with v i r u s and cor re la ted s u r v i v a l with the time i t took c a t e r p i l l a r s to eat the t reated d i e t . 14 CHAPTER ONE The Comparative Bio logy and S u s c e p t i b i l i t y of Cabbage Looper (T r ichop lus ia n i ) Populat ions to the Singly-Embedded Nuclear Polyhedrosis V i r u s of T. n i Both the molecular b io logy and ecology of nuclear polyhedrosis v i ruses (NPVs) must be explored to ascer ta in t h e i r po ten t ia l as b i o i n s e c t i c i d e s (Godfray 1995). Knowledge of the molecular b io logy of NPVs has grown considerably in the past 25 years . The i n f e c t i o n pathway of the archetype NPV, Autographa c a l i f o r n i c a NPV (AcNPV), i s f a i r l y wel l understood (Granados and Lawler 1981, Keddie et a l . 1989, B l i s s a r d and Rohrmann 1990, Volkman and Keddie 1990, Engelhard et a l . 1994), the complete genome of AcNPV has been sequenced (Ayres et a l . 1994) and the funct ion of severa l v i r a l genes has been e luc idated (e .g . Carson et a l . 1988, O ' R e i l l y and M i l l e r 1989, B l i s s a r d and Rohrmann 1990, Lu and Carstens 1991, O ' R e i l l e y 1995). In a d d i t i o n , severa l recombinant NPVs have been engineered to increase the speed of k i l l ( e .g . Hammock et a l . 1990, Cory et a l . 1994) and these are awaiting commercial re lease . 15 Conversely, knowledge of the ecology of NPVs i s more l i m i t e d . The ro le of NPVs in the ecosystem i s determined by c h a r a c t e r i s t i c s of the v i r u s and i t s hosts (Fuxa 1987a). Pert inent v i r a l c h a r a c t e r i s t i c s include v i r u l e n c e , host range, t ransmission ra te , pers is tence in the environment and d i s t r i b u t i o n i n space. C h a r a c t e r i s t i c s of host insects include behavior, growth ra te , temporal and s p a t i a l densi ty and d i s t r i b u t i o n . V a r i a t i o n among hosts i n s u s c e p t i b i l i t y to NPVs i s p a r t i c u l a r l y important. The extent to which s u s c e p t i b i l i t y v a r i e s among i n d i v i d u a l s and i s her i t ab le w i l l determine the po ten t ia l for hosts to evolve res is tance to NPVs. The development of res is tance i s a concern i f NPVs are to be used as insect cont ro l agents. Resistance to pathogens or chemicals of ten involves f i t n e s s costs and i s unstable . Lines that were r e s i s t a n t have been shown to rever t to s u s c e p t i b i l i t y when the s e l e c t i v e pressure i s removed (e .g . Fuxa and Richter 1989, Bush et a l . 1993, Cochran 1993, Tabashnik et a l . 1994). In such instances, i t may be p o s s i b l e to prevent or re tard the evolut ion of res is tance by a l te rna t ing the use of i n s e c t i c i d e s with d i f f e r e n t modes of ac t ion ( e . g . Georghiou 1983). The po ten t ia l costs of res is tance to NPVs can be measured by comparing the f i t n e s s of r e s i s t a n t and 16 suscept ib le l i n e s in the absence of v i r u s . In addi t ion to being l e t h a l , NPVs may a f f e c t the populat ion dynamics of the host by adversely in f luenc ing the sex r a t i o and/or q u a l i t y of s u rv iv ors . For example, NPVs may be more in fec t ious to females than males (Doane 1976, Entwist le and Evans 1985) and subletha l i n f e c t i o n s could reduce f i t n e s s in severa l ways: by prolonging development, reducing pupal weight, impair ing reproduction ( fecundity and/or v i a b i l i t y of eggs) and/or causing the wings of surv iv ing adults to be deformed (Va i l and Ha l l 1969, see Rothman and Myers 1996 for a review). The purpose of t h i s chapter was three f o l d : (1) to determine whether the s u s c e p t i b i l i t y of T r i c h o p l u s i a n i to the singly-embedded nuclear polyhedrosis v i r u s of T. n i var ied among 12 populat ions of cabbage loopers . V a r i a t i o n among populat ions would suggest the p o s s i b i l i t y of genet ica l ly -based res is tance (see Boots and Begon 1995). (2) To determine whether t h e . s u s c e p t i b i l i t y of a populat ion was cor re la ted with the f i t n e s s of i n d i v i d u a l s i n v i r u s - f r e e environments. (3) To determine i f the pupal weight, developmental time, sex r a t i o and wing formation of i n d i v i d u a l s surv iv ing exposure to TnSNPV d i f f e r e d from c o n t r o l s . 17 MATERIALS AND METHODS Source of cabbage loopers and laboratory rear ing protoco l I establ ished 12 populat ions of cabbage loopers by obta in ing eggs or pupae from var ious laboratory cu l tures and c o l l e c t i n g larvae from greenhouses and a cabbage (Brass ica oleracea L.) patch (Table 1.1). The 12 populat ions were cul tured in the laboratory at 26+1°C at a photoperiod of a 16:8 (L:D) . Eggs were surface s t e r i l i z e d by soaking them i n a 0.2% sodium hypochlor i te s o l u t i o n for 3 min, r insed in d i s t i l l e d water for 3 min and a i r d r i e d . For each popula t ion , 10 neonates were placed in a 300 ml styrofoam cup contain ing 25 ml of high wheat germ d ie t (Table 1.2) , with 25 cups per popula t ion . Pupae were c o l l e c t e d , washed in 0.6% sodium hypochlor i te for 10 min, then r insed i n d i s t i l l e d water fo r 10 min. Surv iva l to the pupal stage was >90 % for a l l populat ions. Pupae (70-75) were placed i n c y l i n d r i c a l cages (Ignoffo 1963), wrapped with paper toweling as an o v i p o s i t i o n subst ra te . A b o t t l e containing 10% sucrose s o l u t i o n and a cotton wick was provided as food for adu l ts . Eggs were c o l l e c t e d every second day a f te r females s tar ted lay ing and placed at 4 *C to a r r e s t embryonic development. Af ter the four th c o l l e c t i o n , eggs were 18 surface s t e r i l i z e d as prev ious ly described and the cyc le was repeated. I cu l tured a l l 12 populat ions fo r >_ 5 generations to accl imate them to my rear ing protocol p r i o r to t e s t i n g . Determination of the s u s c e p t i b i l i t y of cabbage looper populat ions to TnSNPV The inoculum of TnSNPV used in the fo l lowing assays was obtained from Dr. R. P. Jaques (Agr icu l ture and A g r i -Foods Canada, Harrow, Ontar io , Canada). This sample of v i r u s had been i s o l a t e d from diseased larvae of the Jaques populat ion according to the protocol of Potter et a l . (1978J. I quant i f i ed the s o l u t i o n 10 times (mean = 2.565 x 10 occ lus ion bodies [OBs]/ml) using an improved Neubauer hemacytometer (0.01 mm deep) with br ight l i n e under phase-contrast microscopy at 450X. The sample was s e r i a l l y d i l u t e d i n s t e r i l e d i s t i l l e d water to 2.565 x 10 b , 1.283 x 10 f e, 2.565 x 10 s , 1.283 x 10* and 2.565 x 10** OBs/ml with each d i l u t i o n being r e p l i c a t e d 6 t imes. Immediately fo l lowing the f i r s t c o l l e c t i o n , eggs from each of the 12 populat ions were surface s t e r i l i z e d . A f te r hatch, neonates were placed i n d i v i d u a l l y i n 25 ml p l a s t i c cups containing high wheat germ d i e t . At 168 hr post -hatch ( f i f t h i n s t a r ) , larvae were t ransfer red i n d i v i d u a l l y to a 19 second cup containing only a plug of d ie t ( thickness = 2-3 nun, diameter = 5 mm) treated with d i s t i l l e d water (contro ls ) or 25650, 12830, 2565, 1283 or 257 TnSNPV OBs (10 jal of one of the above d i l u t i o n s ) . Each dose was r e p l i c a t e d 6 times with 7 larvae per r e p l i c a t e . Cohorts of 18-25 larvae were used as contro ls depending on a v a i l a b i l i t y . Larvae that consumed the en t i re plug wi th in 24 hr (97%: r e s u l t i n g r e p l i c a t e s i z e = 6-7 larvae) were returned to t h e i r o r i g i n a l cup while those f a i l i n g to do so were d iscarded. Larvae were checked d a i l y and mor ta l i t y , pupal weight (on day of pupat ion) , time of adult emergence, sex and wing condi t ion (see V a i l et a l . 1969) of moths were recorded. Diagnosis of v i r a l mor ta l i ty was based on gross symptoms. O v e r a l l , 94% of TnSNPV inoculated larvae that d ied exhib i ted the t y p i c a l s igns of nucleopolyhedrosis i . e . d i s c o l o r a t i o n and l i q u e f a c t i o n (Evans and Entwist le 1987). The equation of the dosage-mortal i ty curves and LD50s (dose required to k i l l 50% of t reated ind iv idua ls ) with associated 95% confidence i n t e r v a l s were computed for each T. n i populat ion using a p rob i t procedure developed by the S t a t i s t i c a l Ana lys is System (SAS 1990). In most populat ions, cont ro l mor ta l i ty was 0% and i t exceeded 5% for only one populat ion (8%: 2/25). Thus, the data used in the prob i t analyses (see Finney 1971) were not corrected for cont ro l 20 morta l i t y . Slopes and in tercepts of regressions for the 12 populat ions were compared as descr ibed by C o l l e t t (1991). B r i e f l y , t h i s involved f i t t i n g the fo l lowing three p rob i t models. The f i r s t f i t t e d model constrained a l l populat ions to have the same slope and in te rcep t . The second model f i t t e d a common slope but allowed in tercepts to vary among populat ions. The t h i r d model allowed both the slope and in tercept to vary among popula t ions . The d i f fe rence between the deviance (2 x l o g - l i k e l i h o o d ; C o l l e t t 1991) of the t h i r d and second model has a ch i -square d i s t r i b u t i o n under the n u l l hypothesis that the slopes are i d e n t i c a l . A large change in deviance therefore impl ies that the slopes are d i f f e r e n t . The degrees of freedom are given by the d i f fe rence i n the number of parameters estimated in the t h i r d model minus the second one. Here, 24 parameters (12 slopes + 12 in tercepts) were estimated in the t h i r d model, 13 parameters (1 slope and 12 in tercepts) in the second and thus there were 11 degrees of freedom. S i m i l a r l y , the d i f fe rence between the deviance of the second and f i r s t model can be used to tes t for equa l i ty of in te rcep ts . Degrees of freedom were again 11 s ince 13 parameters were estimated i n the second model and 2 i n the f i r s t . 21 Sublethal e f f e c t s of TnSNPV The poss ib le subletha l e f f e c t s of TnSNPV on the durat ion of the l a r v a l and pupal stages and pupal weight were examined using analyses of var iances (PROC GLM, SAS 1990). The independent va r iab les were sex (2 l e v e l s , f i xed e f f e c t ) , TnSNPV dose (5 doses + 1 cont ro l = 6 l e v e l s , random e f fec t ) and T. n i populat ion (12 l e v e l s , random e f f e c t ) . Examination of res idua ls d id not suggest that the assumptions of ANOVA were v i o l a t e d . Data were pooled across r e p l i c a t e s wi th in a TnSNPV dose. Contigency tables were used to determine i f the sex r a t i o and the incidence of deformed wings of the surv ivors var ied among (1) the 6 experimental groups (1 cont ro l and 5 TnSNPV doses) and (2) the cont ro l and in fected ( i . e . the 5 TnSNPV doses pooled) groups. Furthermore these analyses were conducted on each cabbage looper populat ion and for the 12 populat ions combined. F i tness of cabbage looper populat ions in the absence of TnSNPV To determine i f res is tance to TnSNPV i s c o s t l y , the mean durat ion of the l a r v a l and pupal stages, pupal weight, egg production and % egg hatch (a rcs in \ /% egg hatch transformed) 22 (Pearson, 1 - t a i l p r o b a b i l i t y ) to the LD50 of t h e i r populat ion. The number of cont ro l p a i r s var ied from 8 to 11 per populat ion because (1) there i n i t i a l l y were fewer contro l i nd iv idua ls in some populat ions and of (2) uneven sex r a t i o . Hence, for sake of consistency, the number and v i a b i l i t y of eggs l a i d by cont ro l females were estimated by mating the f i r s t 8 pa i rs of adults to emerge from the contro l group of each populat ion in 500 ml paper cups ( i . e . one pa i r / cup and 8 cups/popula t ion) . The ins ide of each cup was l i n e d with a p iece of paper toweling as o v i p o s i t i o n substrate and a 20 ml v i a l containing a 10% sucrose so lu t ion with a wick was provided for adult feeding. The f i r s t paper l i n i n g was c o l l e c t e d 4 days a f te r a pa i r was formed and every second day thereaf ter u n t i l the female d i e d . The number of eggs and percent eggs hatching per l i n i n g were counted. In these analyses, the F e d e r i c i and Sandoz populat ions were pooled because they share a common ancestry (Table 1.1), and thus the LD50 and mean l i f e - h i s t o r y t r a i t s were based on the combined data s e t s . The Jaques and Kaupp populat ions were pooled for the same reasons (Table 1.1) . Hence, the Pearson cor re la t ions were based on N = 10 cabbage looper populat ions. 23 RESULTS Determination of the s u s c e p t i b i l i t y of cabbage looper populat ions to TnSNPV The slopes of the 12 dosage-mortal i ty curves were not s i g n i f i c a n t l y d i f f e r e n t (chi -square = 0.20, df = 11, P > 0.50; Table 1.3) but some intercepts d i f f e r e d (chi -square = 49.99, df = 11, P < 0.01; Table 1.3) . There was a 3 . 5 - f o l d d i f fe rence between the lowest LD50 (6465 OBs - Hughes populat ion) and highest LD50 (22395 OBs Keddie populat ion; Figure 1.1). However, 8 of 12 LD50s var ied between 8225-13947 OBs and, i n most cases 95% confidence i n t e r v a l s overlapped (Figure 1.1). The only exceptions were the Hazelmere, Keddie and Langley populat ions that had s i g n i f i c a n t l y higher LD50s than Hughes, Sandoz, F e d e r i c i or Jaques (Figure 1.1). The Sandoz and F e d e r i c i populat ions are re la ted (Table 1.1) and t h e i r LD50s were near ly i d e n t i c a l , being 8225 and 8380 OBs, respec t i ve ly (Figure 1.1). The Jaques and Kaupp populat ions are a lso re la ted (Table 1.1) and t h e i r LD50s were not s i g n i f i c a n t l y d i f f e r e n t (Figure 1.1). Two of three populat ions with high LD50s (»^20000 OBs; Hazelmere and Langley) had been es tab l ished from ind iv idua ls recent ly c o l l e c t e d from the wi ld (greenhouses; Table 1.1). 24 Sublethal e f f e c t s of TnSNPV on the developmental t ime, pupal weight, sex r a t i o and wings of surv ivors The durat ion of both the l a r v a l and pupal stages were independent of TnSNPV dose but d i f f e r e d between sexes (females had shorter l a r v a l and pupal stages than males) and among T. n i populat ions (Table 1.4 and Figures 1.2 and 1.3). Female pupae were s i g n i f i c a n t l y l i g h t e r than those of males (Table 1.4 and Figure 1.4). The s i g n i f i c a n t dose*populat ion in te rac t ion term suggests that the e f f e c t of dose on pupal weight var ied among T. ni. populat ions (Table 1.4 and Figure 1.4). TnSNPV dose was s i g n i f i c a n t in only 5 of 24 cases when 1-way ANOVAs and Tukey mul t ip le range t e s t s were conducted for each sex of each populat ion (2 sexes x 12 populat ions = 24 analyses; Table 1.5). The pupal weight of females was a f fec ted most o f ten , but the e f f e c t of dose was incons is tent (Table 1.5 and Figure 1.4). The sex r a t i o and incidence of deformed wings among surv ivors i n the 6 treatment groups (1 cont ro l and 5 TnSNPV doses) d i d not d i f f e r whether the 12 populat ions were analysed separately or combined (chi -square ana lys is of 2 x 6 contigency tab le ; sex r a t i o , a l l P 's > 0.22; deformed wings, a l l P 's > 0.10). There a lso was no d i f fe rence when the 5 v i r u s concentrat ions were pooled and compared to cont ro ls 25 regardless of whether populat ions were invest igated separately or combined (chi -square analyses of 2 x 2 contigency tab le with Yates' c o r r e c t i o n ; sex r a t i o a l l P 's > 0.10; deformed wings; a l l P 's > 0.10). F i tness of cabbage looper populat ions i n the absence of v TnSNPV Mean egg production per contro l female was s i g n i f i c a n t l y lower i n populat ions with high LD50s (Table 1.6). However, mean egg hatch (a rcs in \j% transformed) and pupal weight of contro l males and females were independent of LD50 of that populat ion (Table 1.6). The mean durat ion of the l a r v a l and pupal stages of both sexes were not cor re la ted with LD50 and the trends were even contrary to those expected (Table 1.6). That i s , the l a r v a l and pupal stage of males and females from populat ions with high LD50s were shorter than those from more suscept ib le populat ions (Table 1.6). Since TnSNPV treatment d id not a f fec t T. n i development (see previous s e c t i o n ) , the mean durat ion of the l a r v a l and pupal stages were r e -estimated and based on a l l surv iv ing i n d i v i d u a l s . The la rger sample s i zes may provide more accurate estimates of these l i f e - h i s t o r y parameters. The four re la t ionsh ips were again negative and not s i g n i f i c a n t (Table 1.6). The mean pupal weight of males and females were s t i l l independent of LD50 26 when the averages were based on a l l surv ivors (Table 1.6). 27 DISCUSSION V a r i a t i o n among populat ions of T. n i i n s u s c e p t i b i l i t y to TnSNPV Resistance to v i r a l i n s e c t i c i d e s could evolve i f the t r a i t i s (1) va r iab le and (2) h e r i t a b l e . S u s c e p t i b i l i t y to TnSNPV var ied among T. n i populat ions and the LD50 of Hazelmere, Keddie and Langley was approximately 20000 TnSNPV OBs compared to about 8000 OBs for F e d e r i c i , Hughes, Jaques and Sandoz. The length of time a populat ion had been cu l tured i n the laboratory d i d not appear to be a strong determinant of LD50. Hazelmere and Langley were the most recent populat ions and t h e i r LD50s were among the three h ighest . However, Abbotsford and Fournier were es tab l ished at the same time and the i r LD50s were s i m i l a r to o lder populat ions. The LD50 of the Keddie populat ion was the second highest and i t had been cul tured in the laboratory fo r at l eas t 10 years p r i o r to t h i s study. V a i l and Tebbets (1990) a lso observed that the length of time under co lon iza t ion d id not a f f e c t the s u s c e p t i b i l i t y of P l o d i a in t e rpunc t e l l a to a granulos is v i r u s . S u s c e p t i b i l i t y to NPVs and to other baculoviruses has been shown to vary among populat ions in P i e r i s brass icae (David and Gardiner 1965), Bombyx mori (Aratake 1973), PI . i n te rpunc te l l a (Hunter and Hoffmann 1973, V a i l and Tebbets 28 1990, Boots and Begon 1995), Epiphvas postv i t tana (Geier and Br iese 1979), Phthorimaea o p e r c u l e l l a (Br iese and Mende 1981) and Spodoptera f rugiperda (Fuxa 1987b). The s i m i l a r LD50s of re la ted populat ions of T. n i suggests that the s u s c e p t i b i l i t y of cabbage loopers to TnSNPV may be g e n e t i c a l l y determined. The F e d e r i c i and Sandoz populat ions share a common ancestry and t h e i r LD50s were near ly i d e n t i c a l . The Jaques and Kaupp populat ions are a lso re la ted and t h e i r LD50s d i d not d i f f e r . Larva l res is tance to NPVs i s cont ro l l ed by one or more genes lack ing dominance in S. f rugiperda (Reichelderfer and Benton 1974), to a s i n g l e dominant autosomal gene in Ph. o p e r c u l e l l a (Briese 1982) and i s polygenic i n B. mori (Aratake 1973). Although the mode of inher i tance i s unknown, res is tance to granulos is v i ruses in P i . brassicae (David and Gardiner 1965) and P_l. i n t e r p u c t e l l a (Hunter and Hoffmann 1973) a lso appears to be g e n e t i c a l l y determined. Br iese and Mende (1983) and Fuxa et a l . (1988) s u c c e s s f u l l y se lec ted for res is tance to NPVs in laboratory populat ions of Ph. o p e r c u l e l l a and S. f rug iperda , r e s p e c t i v e l y . In nature, the po ten t ia l for T. n i larvae to evolve res is tance to TnSNPV could be even greater than suggested by t h i s study. F i r s t , in t rapopulat ion v a r i a t i o n was not 29 considered. Within a given populat ion , s u s c e p t i b i l i t y may vary between i n d i v i d u a l s and t h i s may provide add i t iona l genet ic var iance for natura l s e l e c t i o n . Second, s u s c e p t i b i l i t y to NPV may be more v a r i a b l e i n the w i l d . Founder events, bott lenecks and inbreeding, common in laboratory rea r ing , may have reduced i n t r a - and in terpopulat ion v a r i a t i o n in s u s c e p t i b i l i t y . F i tness of cabbage looper populat ions i n the absence of TnSNPV The mean durat ion of the l a r v a l and pupal stages and mean pupal weights of cabbage loopers were independent of the LD50 of t h e i r populat ion. Resistance to baculovi rus adversely a f fec ted developmental time in PI. i n t e r p u n c t e l l a (Va i l and Tebbets 1990, Boots and Begon 1995) whereas r e s i s t a n t S. f rugiperda developed more r a p i d l y than suscept ib le conspec i f i cs (Fuxa and Richter 1989). Resistance adversely a f fec ted pupal weight in PI. i n te rpunc te l l a (Boots and Begon 1995) but not in S. f rugiperda (Fuxa and Richter 1989). The number of eggs l a i d by cont ro l T. n i females was negat ive ly cor re la ted to the LD50 of t h e i r populat ion suggesting that res is tance to TnSNPV may involve reproduct ive c o s t s . Th is may a lso be the case for S. f rugiperda (Fuxa and 30 Richter 1989) and PI., in terpuncte l l a (Boots and Begon 1995) and t h e i r baculov i ruses . In both spec ies , r e s i s t a n t females l a i d and hatched fewer eggs than s u s c e p t i b l e s . S. f rugiperda res is tance was unstable and was l o s t wi th in one generat ion when exposure to v i r u s was discont inued (Fuxa and Richter 1989). Resistance to chemical i n s e c t i c i d e s and to B a c i l l u s thur ing iens is has f requent ly been shown to incur f i t n e s s costs and to be unstable ( e .g . Bush et a l . 1993, Cochran 1993, Tabashnik et a l . 1994). The po ten t ia l costs of res is tance to TnSNPV i n T. n i suggests that i t may be poss ib le to preclude or slow the evo lu t ion of res is tance by using TnSNPV i n r o t a t i o n with other i n s e c t i c i d e s . The success of ro ta t ions i s based on the assumption that i n d i v i d u a l s r e s i s t a n t to i n s e c t i c i d e A w i l l have lower f i t n e s s than suscept ib les in the presence of i n s e c t i c i d e B. The i n s e c t i c i d e s used in ro ta t ions should have d i f f e r e n t modes of ac t ion to minimize the p o s s i b i l i t y of c r o s s - r e s i s t a n c e (Georghiou 1983, Tabashnik 1989). Fuxa and Richter (1990) showed that res is tance to S. f rugiperda nuclear polyhedrosis v i r u s conferred c r o s s - r e s i s t a n c e to Autographa c a l i f o r n i c a nuclear polyhedrosis v i r u s and to S. f rugiperda granulosis v i r u s . However, they a lso showed that r e s i s t a n t S. f rugiperda were more suscept ib le to methyl parathion and equal ly suscept ib le to B. thur ing iens is than 31 c o n t r o l s . Resistance to chemical i n s e c t i c i d e s d id not a f f e c t the s u s c e p t i b i l i t y of H e l i o t h i s v i rescens to NPVs (Ignoffo and Roush 1986). Hence, an integrated pest management scheme, using chemical i n s e c t i c i d e s , B. thur inq iens is and TnSNPV in ro ta t ion may provide e f f i c i e n t cont ro l of T. n i while minimizing the p o s s i b i l i t y of cabbage loopers evolv ing res is tance to any of these s e l e c t i n g agents. Sublethal e f f ec ts of TnSNPV on T. ni The e f f e c t of TnSNPV on cabbage loopers inoculated at f i f t h ins ta r appears to be r e s t r i c t e d to mor ta l i t y . Indiv iduals surv iv ing exposure to the v i r u s were s i m i l a r to contro ls in the durat ion of the l a r v a l and pupal stages, wine condi t ion and sex r a t i o . V a i l and H a l l (1969) a lso showed that exposure to TnSNPV d id not a f fec t the durat ion of the l a r v a l stage or wing condi t ion of surv iv ing cabbage looper adu l ts . Male-biased sex r a t i o s fo l lowing NPV ep izoo t ics have been observed in L. d ispar (Campbell 1963b), Neodiprion s e r t i f e r (Bird 1961), and N. p r a t t i p r a t t i (Mclntyre and Dutky 1961). Females are be l ieved to be more suscept ib le to NPVs in these species because they require more t ime / ins ta rs than males to pupate. In cabbage loopers , both sexes have f i v e ins ta rs and the l a r v a l stage of females i s only s l i g h t l y shorter (0.5 day at 26°C) than males. 32 V i r a l i n f e c t i o n had no consistent e f f ec t on the pupal weight of surv iv ing T. n i . TnSNPV dose was s i g n i f i c a n t in only 5 of 12 populat ions. For 3 of these 5 populat ions, pupal weight d i f f e r e d in only 1 of 15 poss ib le pairwise comparisons of doses. The e f fec t of dose was most consis tent in the Sandoz populat ion but i t i s d i f f i c u l t to i n t e r p r e t . Female pupae surv iv ing the middle dose (2565 OBs) were heavier than those surv iv ing the two highest or lowest doses. Ignoffo (1964) and V a i l and Ha l l (1969) a lso showed that pupal weight was independent of TnSNPV treatment. Pupal weight i s a cor re la te of fecundity i n many species inc lud ing T. n i and thus, t h i s study supports the f ind ings of V a i l and H a l l (1969) that subletha l TnSNPV i n f e c t i o n s do not a f f e c t the reproductive success of cabbage loopers . The f ind ings of t h i s chapter are three f o l d . F i r s t , the s u s c e p t i b i l i t y of cabbage loopers to TnSNPV var ied among populat ions of T .^ n i . Second, cont ro l females from populat ions with high LD50s produced fewer eggs than females from more suscept ib le populat ions suggesting that res is tance to TnSNPV may involve a c o s t . T h i r d , the e f f e c t of TnSNPV on the demography of cabbage loopers appears to be r e s t r i c t e d to d i r e c t mor ta l i ty as ind iv idua ls surv iv ing exposure to TnSNPV seemed, at l eas t in terms of developmental time and pupal 3 3 weight, to be s i m i l a r to cont ro ls 34 Table 1.1 H i s t o r y of c a b b a g e l o o p e r p o p u l a t i o n s . Designation Source Abbotsford MLM collected approx. 150 larvae from cabbage plot near Abbotsford B.C. in 1992. (AB) Several applications of chemical insecticides prior to collection. Arizona Eggs from USDA-ARS, Phoenix, Az. in 1992. Source established ca. 1970 from insects (AZ) collected in Arizona. Federici Eggs from Dr. B. Federici, UC Riverside in 1992. Source established in 1968 from insects (FE) collected near Irvine, Ca. Fournier Eggs from Mr. F. Fournier, Agr. Canada, St-Jean, Qc. in 1992. Source established in (FO) 1992 from insects collected from cabbage plot near Montreal, Qc. Hazelmere MLM collected approx. 100 larvae from cucumber plants in Hazelmere greenhouse, (HA) Langley, B.C. in 1992. Several applications of Bacillus thuringiensis prior to collection. Hughes Eggs from Dr. P. Hughes, Boyce-Thompson Institute, Ithaca, NY in 1991. (HU) Source established in 1976. Ignoffo Eggs from Dr. C. Ignoffo, USDA-ARS, Columbia, Mo. in 1991. Source established in 1965 (I) from insects collected in Brownsville, Tx. Jaques Eggs from Dr. R. Jaques, Agr. Canada, Harrow, On. in 1990. Source established in 1958 (J) from culture at Geneva Experiment Station, Geneva, NY. Kaupp Eggs from Dr. W. Kaupp, Forestry Canada, Sault-Ste-Marie, On. in 1991. Source (KA) established in 1985 from Jaques population. Keddie 54t£ 32? pupae from Dr. A. Keddie, U. of Alberta, Edmonton, Ab. Source established in (KE) 1982 from cultures of Dr. L. Caltagirone (UC Berkeley), Chevron Chemicals (Richmond, Ca) and Dow Chemicals (Ca). Langley MLM collected approx. 100 larvae from green pepper plants in Langley (L) greenhouse, Langley, B.C. in 1992. Several applications of Bacillus thuringiensis prior to collection. Sandoz Eggs from Sandoz Crop Protection Co., Wasco, Ca. in 1992. Source established in 1973 (S) from Federici population. 3 5 Table 1.2 D ie t f o r m u l a t i o n for c a b b a g e l o o p e r l a r v a e . Ing red ient A m o u n t (g) W h e a t g e r m 4 2 0 C e l l u l o s e 2 5 2 W e s s o n sa l t m i x 8 4 C a s e i n 2 9 4 S u c r o s e 1 5 4 C h o l e s t e r o l 2 5 A s c o r b i c a c i d 2 8 S o d i u m a l g i n a t e 4 2 S o r b i c a c i d 1 0 A l f a l f a m e a l 8 0 V a n d e r z a n t v i t a m i n m i x 1 2 8 S t r e p t o m y c i n s u l f a t e 0 . 5 F o r m a l i n e 2 . 4 D i s t i l l e d w a t e r 4 5 0 0 A g a r 1 0 0 D i s t i l l e d w a t e r 3 0 0 0 36 Table 1.3 C o m p a r i s o n of t h e s l o p e s a n d i n t e r c e p t s of t h e d o s a g e - m o r t a l i t y c u r v e s of t h e 12 c a b b a g e l o o p e r p o p u l a t i o n s . MODEL INTERCEPT SLOPE NO. DEVIANCE PARAMETERS ESTIMATED I. Common slope/ common intercept II. Common slope/ 1 intercept for each population AB AZ FE FO HA HU I J KA KE' L S III. 1 slope/ 1 intercept for each population AB AZ FE FO HA HU I J KA KE L S -8.57 -8.70 -8.86 -8.39 -8.73 -9.26 -8.18 -8.86 -8.44 -8.75 -9.28 -9.32 -8.42 -9.08 -10.62 -7.02 -7.13 -12.0 -7.31 -7.89 -8.44 -9.62 -8.17 -11.59 -9.32 2.10 2.15 2.24 2.59 1.79 1.74 2.81 1.92 1.90 2.15 2.37 1.88 2.69 2.38 -2482.24 -2432.25 13 -2432.05 24 3 7 38 Table 1.4 C o n t i n u e d . S o u r c e P u p a l W e i g h t df M S F P S e x 1 1 3 8 1 5 1 6 0 1 6 7 . 9 <0 .001 P o p u l a t i o n 11 3 5 5 7 2 2 4 . 3 <0 .001 D o s e 5 1 7 9 5 7 8 2 . 2 0 . 0 5 S e x * P o p u l a t i o n 11 6 0 7 4 6 0 . 7 0 . 7 0 S e x * D o s e 5 9 5 8 5 4 1.2 0 . 3 2 D o s e * P o p u l a t i o n 5 5 1 5 4 8 0 6 1.9 <0 .001 S e x * D o s e * P o p u l a t i o n 5 5 7 3 2 0 0 0 . 9 0 . 7 0 39 Table 1.5 T n S N P V d o s e ( n u m b e r of O B s ) w h i c h r e s u l t e d in s i g n i f i c a n t v a r i a t i o n in p u p a l w e i g h t of i n d i v i d u a l s s u r v i v i n g to a d u l t h o o d b y t h e T u k e y mu l t ip le r a n g e test . P o p u l a t i o n S e x R e s u l t s F e d e r i c i F o u r n i e r K e d d i e L a n g l e y S a n d o z F e m a l e F e m a l e M a l e F e m a l e F e m a l e 12825>257, 12825>0 257>0 257>12825 25650>257 2565>0, 2565>57, 2565>12825, 2565>25650 40 Table 1.6 P e a r s o n ' s r (1 - ta i l p robab i l i t y ) r e l a t i n g m e a n l i f e - h i s t o r y t ra i ts of m a l e s a n d f e m a l e s to t h e L D 5 0 of the i r l ine (N=10) . C h a r a c t e r i s t i c s C o n t r o l s O n l y A l l I n d i v i d u a l s E g g s l a i d / f e m a l e - 0 . 5 9 (0.04) A r c s i n ^ / % " e g g h a t c h 0 . 0 3 (0.47) D u r a t i o n of l a rva l s t a g e F e m a l e - 0 . 3 4 ( 0 . 1 7 ) - 0 . 4 6 ( 0 . 0 9 ) M a l e - 0 . 2 4 (0.26) - 0 . 3 5 5 (0 .16) D u r a t i o n of p u p a l s t a g e F e m a l e - 0 . 3 8 ( 0 . 1 4 ) - 0 . 1 0 ( 0 . 3 9 ) M a l e - 0 . 1 7 ( 0 . 3 2 ) - 0 . 3 2 (0 .18) P u p a l w e i g h t F e m a l e 0 . 2 3 (0 .26) 0 . 0 4 (0 .45) M a l e 0 . 2 4 ( 0 . 2 5 ) 0 . 3 8 (0 .14) 41 Figure 1.1 LD50 and associated 95% confidence l i m i t s of each cabbage looper populat ion. 42 o O o O O o o o O o O O o o o O o O O o o LT) O LO o m o IT) CO CO CM CM 1— 1— (sgo) 09Q1 4 3 Figure 1.2 Mean (+1 SE) durat ion of the l a r v a l stage (days) of surv iv ing females (•) and males (A) averaged across a l l TnSNPV doses for each cabbage looper populat ion. 44 - ZOpUBS - Ae|6uB-| - 9!PP9>i ddne>| \- senbep OJ,}OU6| S9L |6nH 8 J 8 L U | 8 Z B H o L O L O o — r ~ L O C O o C O L O c \ i jejiunoj puepej euozuv pjoisjoqqv O c\i (sAep) e6v\s IBAJBI JO uo\veinQ 4 5 Figure 1.3 Mean {±1 SE) duration of the pupal stage (days) of surviving females (•) and males (A) averaged across a l l TnSNPV doses for each cabbage looper population. 46 I C M C O o cd C O C D ^ O J o ZOPUBS A 8 | 6 U B - | 9|PP9>1 - ddne^ - sanbep - OJJOU6| •S8L |6nH - 8 J 8 U J | 8 Z B H - jajujnoj puepa j euozuv pjoisioqqv (sAep) 86BIS |Bdnd p uojiBjna 47 Figure 1.4 Mean (+1 SE) pupal weight (mg) of surv iv ing females (•) and males ( A ) in r e l a t i o n to TnSNPV dose for each cabbage looper populat ion. 48 (6iu) ; L | 6 ! 9 M | B d n d UB9| / \ | 4 9 (6UJ) IL|6!9M rednd UB9|/\| 50 CHAPTER TWO Var i a t i o n i n S u s c e p t i b i l i t y to the Singly-Embedded Nuclear Polyhedrosis Virus of Trichoplusia n i Within a Line of Cabbage Loopers For c a t e r p i l l a r s to evolve resistance to nuclear polyhedrosis viruses (NPVs), s u s c e p t i b i l i t y must be a variable and heritable t r a i t (e.g. Endler 1986). Many studies have shown that the s u s c e p t i b i l i t y of larvae to NPVs varies across populations (see references i n Chapter 1). However, I am unaware of any study that has quantified the amount of genetic v a r i a b i l i t y for s u s c e p t i b i l i t y to NPVs that exists within a population. For obvious reasons, most ecological studies of lepidopterans are conducted on a regional scale. From an applied perspective, i t may also be more relevant to determine the potential of a l o c a l population, for example a population of H e l i o t h i s spp. i n a corn (Zea spp) or cotton (Gossypium spp) f i e l d or a population of Lymantria dispar i n an oak forest (Quercus spp.), to evolve resistance to NPVs. 51 Thus, determining the amount of he r i t ab le v a r i a t i o n for s u s c e p t i b i l i t y to NPVs that e x i s t s wi th in a populat ion may be of greater in te res t to populat ion b i o l o g i s t s and pest managers. The narrow sense h e r i t a b i l i t y of s u s c e p t i b i l i t y to NPVs (h* ), i s h* = Va / Vp (2.1) where Va i s the addi t ive genet ic variance and Vp the phenotypic var iance i n s u s c e p t i b i l i t y of the populat ion studied (Falconer 1989). In Chapter 1, I observed that the s u s c e p t i b i l i t y of cabbage loopers (T r ichop lus ia ni.) to the singly-embedded nuclear polyhedrosis v i r u s of T. ni. (TnSNPV) var ied among populat ions of T. n i . In t h i s chapter, I examined i f s u s c e p t i b i l i t y to TnSNPV a lso va r ied w i th in a popula t ion , and i f i t was h e r i t a b l e . F i r s t , I conducted t h i s experiment with the Jaques popula t ion . I chose t h i s populat ion because i t had been cul tured i n the laboratory fo r near ly 40 years (Table 1.1) and during t h i s time i t had been through many genet ic bott lenecks because of sporadic outbreaks of d isease . Hence, there may be l i t t l e genet ic v a r i a t i o n among i n d i v i d u a l s for 52 s u s c e p t i b i l i t y to TnSNPV in t h i s populat ion (see Falconer 1989). Second, I repeated the experiment with a populat ion that had been estab l ished by mixing i n d i v i d u a l s from a l l 12 T. n i populat ions s tudied i n Chapter 1. This composite populat ion may be g e n e t i c a l l y more d iverse than the Jaques populat ion and w i l l provide an i n t e r e s t i n g comparison. I a lso inbred i n d i v i d u a l s of the composite populat ion to experimental ly determine the e f f e c t of genet ic bott lenecks on the h e r i t a b i l i t y of s u s c e p t i b i l i t y of T. n i to TnSNPV. Measuring the h e r i t a b i l i t v of s u s c e p t i b i l i t y to TnSNPV S u s c e p t i b i l i t y (S; or tolerance according to Finney 1971) to TnSNPV can be def ined as the maximum or threshold dose (T) of v i r u s that a cabbage looper la rva can ingest without succumbing to the d isease . S u s c e p t i b i l i t y to TnSNPV i s l i k e l y to vary among c a t e r p i l l a r s and larvae with low threshold doses w i l l be h igh ly suscept ib le to the v i r u s . Conversely, those with high threshold doses w i l l be weakly s u s c e p t i b l e . Within a given populat ion of T. n i , s u s c e p t i b i l i t y to TnSNPV should fol low a normal d i s t r i b u t i o n (see Finney 1971). It i s not poss ib le to d i r e c t l y measure the s u s c e p t i b i l i t y of cabbage loopers to TnSNPV (see Finney 53 1971). Moreover, although s u s c e p t i b i l i t y w i l l be continuously d i s t r i b u t e d , the observable (phenotypic) v a r i a t i o n in s u s c e p t i b i l i t y w i l l be discont inuous. That i s , cabbage loopers challenged with v i r u s w i l l f a l l into one of two d isc re te categor ies: dead or a l i v e . The threshold model of quant i ta t ive genet ics (Falconer 1989) has frequent ly been used to estimate the h e r i t a b i l i t y of t r a i t s with discontinuous phenotypic v a r i a t i o n (see Roff 1996 for a review) and was used by Tabashnik and Cushing (1989) to measure the h e r i t a b i l i t y of the s u s c e p t i b i l i t y of P l u t e l l a x y l o s t e l l a to the microbia l i n s e c t i c i d e B a c i l l u s t h u r i n g i e n s i s . The threshold model assumes that the outcome of exposure to TnSNPV (m; surv iva l or death) i s cont ro l l ed by an underly ing continuous var iab le (S; s u s c e p t i b i l i t y to the v i rus ) that has a threshold at S = T. Larvae that i n h e r i t a value of S < T die from TnSNPV, whereas those with S >. T are r es is tan t to the v i r u s (at that dose) . B u l l et a l . (1982) showed that the among family variance,QJ, for s u s c e p t i b i l i t y i s ff-0 (2.2) Kl-I 54 where f i s the number of fami l i es and n i s the average number of Tj. n i larvae per fami ly . Furthermore, the mean s u s c e p t i b i l i t y of the populat ion, P, i s f P= -r i\ f i=i (2.3) with p^ being the proport ion of i n d iv id u a ls dying from TnSNPV in the i t h fami ly . The between-family sums of squares, SS, i s f S5 = £(p;-P) (2.4) The sample s t a t i s t i c (n x SS) / [P x (1-P)] has a chi -square d i s t r i b u t i o n with f - 1 degrees of freedom under the n u l l A t hypothesis that Oj= 0. The i n t r a c l a s s c o r r e l a t i o n , the proport ion of v a r i a t i o n among f a m i l i e s , of m can be estimated by (2.5) 6 P f l - P ) When P i s c lose to 0.5, the c o r r e l a t i o n for the underly ing continuous var iab le i . e . s u s c e p t i b i l i t y i s B u l l et a l . 1982 provide a table to d e t e r m i n e ^ f rom when 55 P 4 0 .5. The h e r i t a b i l i t y of the under ly ing continuous var iab le i . e . s u s c e p t i b i l i t y for a f u l l s i b l i n g design i s 2 x JJ . See B u l l et a l . (1982) for a complete de r iva t ion of equations 2.2 - 2.6. Quant i ta t ive genet ic models are appropriate to study the genetic bas is of res is tance to NPVs for two reasons. F i r s t , two of three studies that invest igated the genet ics of res is tance to NPVs concluded that res is tance may be con t ro l l ed by several genes (Aratake 1973, Reichelder fer and Benton 1974 but see Br iese 1982). Second, quant i ta t ive genetic techniques make no assumptions about the number of genes c o n t r o l l i n g a t r a i t and can be used even in the case of monogenic inher i tance . V i a (1986) advocated the use of quant i ta t ive genetic models to study the evolut ion of res is tance to chemical i n s e c t i c i d e s . 56 MATERIALS AND METHODS H e r i t a b i l i t v of s u s c e p t i b i l i t y to TnSNPV of the Jaques populat ion I i n d i v i d u a l l y reared 80 larvae of the Jaques populat ion (Table 1.1) in 25 ml p l a s t i c cups containing high wheat germ d i e t (Table 1.2). The moths were sexed at emergence and randomly pai red in 500 ml paper cups (see Chapter 1) . I c o l l e c t e d the f i r s t l i n i n g of paper 4 days a f te r a p a i r was formed. Each l i n i n g with eggs was i n d i v i d u a l l y surface s t e r i l i z e d as descr ibed in Chapter 1. The dry papers were i n d i v i d u a l l y placed i n 500 ml paper cups and incubated at 26+l*C. Th i r ty -seven p a i r s produced f e r t i l e eggs. I i n d i v i d u a l l y placed 20 neonates from each of these fami l i es in 25 ml p l a s t i c cups containing high wheat germ d i e t and reared them at 2 6 i l ° C at a photoperiod of 16:8 (L:D) . At 168 hr of age, larvae were in fec ted as descr ibed in Chapter 1; 15 c a t e r p i l l a r s were fed plugs contaminated with 12830 OBs of b TnSNPV (=10 p i of 1.283 x 10 OB/ml: same v i r a l preparat ion as used in Chapter 1) and 5 were given plugs t reated with d i s t i l l e d water. Larvae that consumed the en t i re plug wi th in 24 hr were returned to t h e i r o r i g i n a l cup while those f a i l i n g to do so were d iscarded. A maximum of two larvae per family d id not consume the plug wi th in 24 hr . Larvae were checked 57 d a i l y and mor ta l i ty was recorded. Mor ta l i t y of cont ro l insects never exceeded 1 i n d i v i d u a l per family and was less than 5% over a l l f a m i l i e s . H e r i t a b i l i t v of s u s c e p t i b i l i t y of the composite populat ion I took 75 larvae from a populat ion that had been estab l ished 6 generations e a r l i e r by mixing i n d i v i d u a l s from a l l twelve populat ions l i s t e d in Table 1.1. From these c a t e r p i l l a r s , I obtained 34 p a i r s of moths producing f e r t i l e eggs. For each of these f a m i l i e s , I in fec ted 18-24 larvae per family with 12830 OBs of TnSNPV (=10 p.1 of 1.283 x 10* OB/ml) and mock t reated an add i t iona l 5 c a t e r p i l l a r s per family as c o n t r o l s . A maximum of two larvae per fami ly d id not consume the plug wi th in 24 hr . Control mor ta l i ty never exceeded 1 i n d i v i d u a l per family and was less than 5% over a l l f a m i l i e s . I randomly chose 17 fami l i es and inbred one pa i r of the contro l moths per family (however, only 14 p a i r s produced f e r t i l e eggs). I in fec ted 15 larvae from each inbred family with 12830 OBs of TnSNPV (10 u l of 1.283 x 10* OB/ml) as prev ious ly descr ibed (a maximum of 1 l a rva per f ami ly ) . Mor ta l i t y of contro l insects never exceeded 1 i n d i v i d u a l per family and was less than 5% over a l l f a m i l i e s . I repeated the experiment with the composite popula t ion . 58 However, t h i s time I only had 16 p a i r s of adul ts i n the F l and inbred a l l of the fami l i es in the F2 generat ion. I in fec ted 15 larvae per fami ly in both the F l and F2. 5 9 RESULTS Mor ta l i t y caused by TnSNPV var ied considerably among fami l i es of the Jaques populat ion (Table 2 .1) . The genet ic var iance for s u s c e p t i b i l i t y to TnSNPV was s i g n i f i c a n t (P < 0.001) y i e l d i n g a h e r i t a b i l i t y of about 0.3 (Table 2 .1) . In the f i r s t experiment with the composite popula t ion , the among family genet ic variance for mor ta l i ty to TnSNPV was s i g n i f i c a n t fo r both the F l and the inbred F2 generat ion (Table 2 .1) . In both cases, the h e r i t a b i l i t y for s u s c e p t i b i l i t y to TnSNPV was greater than that of the Jaques populat ion (Table 2 .1 ) . Mor ta l i t y was higher and the h e r i t a b i l i t y of s u s c e p t i b i l i t y was lower i n the inbred F2 than in the F l generation (Table 2 .1) . The estimates of h e r i t a b i l i t y fo r the second experiment with the composite populat ion were s i m i l a r to that of the Jaques populat ion (Table 2 .1 ) . Surv iva l of the inbred F2 generation was again lower than in the F l (Table 2 .1) . Inbreeding had a smaller e f f e c t on the h e r i t a b i l i t y of T. ni. to TnSNPV in t h i s experiment. 60 DISCUSSION There was s i g n i f i c a n t genetic variance for the s u s c e p t i b i l i t y of cabbage loopers to TnSNPV i n both the Jaques and composite population. The e f f e c t of mixing T. n i populations on the h e r i t a b i l i t y of s u s c e p t i b i l i t y i s d i f f i c u l t to interpret. In one experiment, the h e r i t a b i l i t y of the composite population was twice as high, but i n the other one i t was s l i g h t l y lower than the Jaques population. Estimates of the h e r i t a b i l i t y of the same character i n the same organism have frequently been shown to vary among experiments (see Falconer 1989). The h e r i t a b i l i t y of s u s c e p t i b i l i t y to TnSNPV of the composite population may have varied between experiments because of a subtle change i n the environment. However, t h i s would appear u n l i k e l y as the c a t e r p i l l a r s were treated i d e n t i c a l l y , fed the same die t and kept at the same temperature and photoperiod. Moreover, mortality was approximately the same i n both experiments (52% vs 45% for the FI; Table 2.1). A l t e r n a t i v e l y , the h e r i t a b i l i t y of the composite population may have varied between experiments because an atyp i c a l sample of individ u a l s was used i n one of them. Thus, the estimate of h e r i t a b i l i t y from the f i r s t experiment may be more r e l i a b l e since i t was based on a larger number of families and more c a t e r p i l l a r s per family (Table 2.1). However, the conservative approach 61 would be to use the mean of the two experiments. Thus, the h e r i t a b i l i t y for s u s c e p t i b i l i t y of the composite populat ion i s (0.593 + 0.223) / 2 = 0.408. This i s s l i g h t l y higher than that of the Jaques populat ion. Thus, i t appears that mixing populat ions only had a marginal e f f e c t on the among fami ly genet ic v a r i a t i o n in s u s c e p t i b i l i t y to TnSNPV. The e f f e c t of inbreeding on the h e r i t a b i l i t y of s u s c e p t i b i l i t y i s a lso d i f f i c u l t to assess . In the f i r s t experiment, the estimate of h e r i t a b i l i t y dec l ined considerably fo l lowing inbreeding (0.593 to 0.437). However, only a subset of the F l f ami l i es were inbred . In the second experiment with the composite populat ion , a l l f ami l i es were inbred and the h e r i t a b i l i t y of the inbred F2 was near ly i d e n t i c a l to that of the F l . Inbreeding i s expected to i n f l a t e the estimates of h e r i t a b i l i t y , but the e f f e c t i s usua l ly small (Falconer 1989). The h e r i t a b i l i t y for s u s c e p t i b i l i t y to TnSNPV of the Jaques populat ion and the mean of the estimates of the composite populat ion were about 0.3 - 0.4. These values are s l i g h t l y higher or comparable to the estimates of the to lerance of P. x y l o s t e l l a to B. thur inq iens is (0.20 Tabashnik and Cushing 1989) and that of Culex to the chemical i n s e c t i c i d e temephos and permethrin (0.404 and 0.389, 6 2 respectively; F e r r a r i et a l . 1982 i n Roush and Daly 1990). Mousseau and Roff (1987) surveyed 1120 estimates of h e r i t a b i l i t y and found that the mean h e r i t a b i l i t y of morphological characters was 0.51, that of behavioral t r a i t s was 0.37 and the average h e r i t a b i l i t y f or l i f e - h i s t o r y parameters ( t r a i t s related to fitness) was 0.25. F u l l s i b l i n g designs, as the one used i n t h i s experiment, provide an upper estimate of h e r i t a b i l i t y (Falconer 1989). This i s because the covariance i s i n f l a t e d by dominance variance and environmental variance among fam i l i e s . By rearing a l l larvae and adults under the same conditions and rearing/infecting the c a t e r p i l l a r s i n d i v i d u a l l y , I minimized environmental variance (Tabashnik and Cushing 1989). My estimates of h e r i t a b i l i t y w i l l thus be mostly influenced by dominance variance. Mousseau and Roff (1987) showed that nonadditive genetic components of variance are usually very small and that f u l l s i b designs overestimate h e r i t a b i l i t y by less than 10%. Perhaps, more importantly, these estimates of h e r i t a b i l i t y were derived i n the laboratory. T y p i c a l l y , h e r i t a b i l i t y i s lower i n the f i e l d presumably because the environmental variance i s greater (e.g. B u l l 1982, Janzen 1992, Simmons and Roff 1994 and references therein). Simmons 6 3 and Roff (1994) compared the estimates of h e r i t a b i l i t y for morphological t r a i t s and developmental time of f i e l d c r i c k e t s (Gryllus pennsylvanicus) reared i n the laboratory and i n the wild. A l b e i t lower, measurements of h e r i t a b i l i t y were s t i l l substantial i n the wild. Simmons and Roff (1994) concluded that the poten t i a l of that population for evolutionary change as measured i n the laboratory was a reasonable estimate of the potential evolutionary change expected for that population i n the wild. The findings of t h i s chapter suggest that s u s c e p t i b i l i t y to TnSNPV varied within a population of cabbage loopers and that part of t h i s phenotypic variance i s ge n e t i c a l l y determined. These findings suggest that populations of T. n i that are exposed to TnSNPV could evolve resistance to the v i r u s . 64 CD CL >. "S c 0 E c CD 2 CD CL c CO CD £ 1 CO *+— 0 Q . CD CO 03 CD E c c co CD E CO CD I CO CD -O E CM fl) .£} CO I-CD O c CO CO > o —1 CD c CD CO o w -*—• CO -•—» CO CD i— CO cr co o c CO JO. - Q CO —' CD .c CD CO CO CD E CO CD ^ co 15 3 CO W O • i— CM CD CD CO CO " c o _C0 CL o Q_ oq o o d v co co CD CM CO CO CM i n m CO co o cn CO co o o o d < ° v r -_co CO co o o d v co" cn cn CM m m CM CM O CO o d ^ d V CO V -CM . co ^r cn m ^r o o co cn • CD o i-co m CM i -CO CO CD CT CO O co O CL E o O c CD E CD D_ X L U TJ CD i _ .O •C CM CM CM O CM CM CO m r -co i n CM c CD E CD CL X L U TJ S> - Q _ C i - CM 65 CHAPTER THREE Keeping Up with the Red Queen: Cabbage Looper - Nuclear Polyhedrosis V i r u s Arms-Race Just at t h i s moment, somehow or other, they began to run. A l i c e never could quite make out, i n thinking i t over afterwards, how i t was that they began: a l l she remembers i s , that they were running hand i n hand, and the Queen went so fast that i t was a l l she could do to keep up with her: and s t i l l the Queen kept crying "Faster! Faster!" ... The most curious part of the thing was, that the trees and the other things round them never changed t h e i r places at a l l : however, fast they went, they never seemed to pass anything. "I wonder i f a l l things move along with us?" thought poor puzzled A l i c e ... "Are we nearly there?" A l i c e managed to pant out ... "Nearly there!" the Queen repeated. "Why, we passed i t ten minutes ago!" ... just as A l i c e was getting quite exhausted, they stopped ... A l i c e looked around her i n great surprise. "Why, I do believe we've been under the same tree the whole time! Everything i s just as i t was !" "Of course i t i s " , said the Queen: "what would you have i t ? " "Well i n our country," sai d A l i c e , s t i l l panting a l i t t l e , "you'd generally get somewhere else i f you ran very fa s t for a long time, as we have been doing." "A slow sort of country!", said the Queen. "Now, here, you see, i t takes a l l the running you can do, just to stay i n the same place. If you want to go somewhere else, you must run twice as fa s t as that!" Excerpts from Through the looking glass, and what A l i c e found there, Lewis C a r r o l l (1880) 66 The coevolut ion of hosts and pathogens has fasc inated evolut ionary b i o l o g i s t s for many years (e .g . Haldane 1949, F l o r 1956, Fenner and R a t c l i f f e 1965, Anderson and May 1979, Lenski and Levin 1985, Henter and V ia 1995). Host-pathogen coevolut ion a lso has p r a c t i c a l importance in d i s c i p l i n e s ranging from epidemiology (Ewald 1994) to the b i o l o g i c a l cont ro l of pest insects (Evans and Entwist le 1987). Host-pathogen coevolut ion has been modeled extensive ly ( e .g . G i l l e s p i e 1975, Anderson and May 1979, 1981, 1982 1983, 1990, May and Anderson 1979, Levin and Pimentel 1981, Bremmerman and P icker ing 1983, Lenski 1984, Lenski and Levin 1985, Levin and Svanborg Eden 1990, Frank 1992, 1993, 1994, Nowak 1992, L i p s i t c h et a l . 1995 see Frank 1996 for a review). These studies reach two conc lus ions . F i r s t , hosts are expected to evolve res is tance to the pathogen with the l e v e l of res is tance being a funct ion of the cost of the r e s i s t a n c e . If uninfected suscept ib le i n d i v i d u a l s have a higher f i t n e s s than r e s i s t a n t i n d i v i d u a l s , the evolut ion of res is tance could be constrained to some intermediate l e v e l . Second, many evolut ionary t r a j e c t o r i e s are poss ib le for the pathogen, and the endpoint w i l l depend on the r e l a t i o n s h i p between the v i ru lence and the transmission rate of the pathogen. 67 The evolution of pathogens i s best understood i n terms of t h e i r i n t r i n s i c reproductive rate, R 0(Anderson and May 1979). R 0 i s the average number of secondary infections produced by one infected i n d i v i d u a l , and i s the quantity that natural s e l e c t i o n w i l l tend to maximize for pathogens (Anderson and May 1979). Rois given by R0= — £ — (3.1) 0+ b + \T ) where ^ i s the transmission rate of the pathogen, o(the rate of disease induced mortality, \f the disease-free mortality rate, |Q the recovery rate of infected individuals and, N the population s i z e of the host (Anderson and May 1979). If the transmission rate and the virulence of the pathogen are independent, then R0 w i l l be maximized at (X = 0 (3 .1) . That i s , s e l e ction w i l l favor avirulence because pathogens that i n f l i c t l i t t l e harm to t h e i r hosts w i l l have higher i n t r i n s i c reproductive rates than more v i r u l e n t ones. In other instances, transmission and host recovery rates may depend on the virulence of the pathogen. For example, v i r u l e n t pathogens capable of causing skin lesions may be transmitted by arthropod vectors more e f f e c t i v e l y than less 6 8 v i r u l e n t ones (Ewald 1994). Here, R©will be maximized by having o(~>o0# and a coevolutionary arms-race (Dawkins and Krebs 1979) may ensue. Pathogens w i l l evolve ever increasing virulence and hosts w i l l keep pace by counter-evolving increased resistance. Van Valen (1973) proposed the Red Queen hypothesis which predicts that the resistance of the host w i l l continuously increase to counteract the increasing virulence of the pathogen. Anderson and May (1983) chose nuclear polyhedrosis viruses (NPVs) as an example of a pathogen for which transmission and virulence are p o s i t i v e l y correlated. C a t e r p i l l a r s contract the disease by ingesting f o l i a g e contaminated with occlusion bodies which contain the v i r a l p a r t i c l e s of NPVs (e.g. Evans and Entwistle 1987). Approximately 7-10 days post-infection, the larvae die, lyse and release occlusion bodies i n the environment that can l a t e r serve as inoculum for other host i n d i v i d u a l s . Selection w i l l thus favour v i r a l genotypes that are s u f f i c i e n t l y v i r u l e n t to k i l l the host larva. However, sel e c t i o n w i l l also concurrently favour c a t e r p i l l a r s resistant to the disease. S u s c e p t i b i l i t y to NPVs has been shown to vary among individuals and to be gene t i c a l l y inherited (Aratake 1973, Reichelderfer and Benton 1974, Briese 1982 see also David and Gardiner 1965, Hunter and Hoffmann 1973, Briese and Mende 69 1983, Fuxa et a l . 1988). Hence, NPVs and lepidopterans both appear to have the c h a r a c t e r i s t i c s necessary for a coevolut ionary arms-race to occur . NPVs a lso o f f e r an environmentally f r i e n d l y a l t e r n a t i v e to chemical i n s e c t i c i d e s as cont ro l agents against pest lepidopterans (see General Introduct ion e . g . Entwist le 1983, Maramorosch and Sherman 1985). Perhaps the s ing le most appealing feature of NPVs i s the p o s s i b i l i t y that a s i n g l e a p p l i c a t i o n of v i r u s could lead to long-term pest c o n t r o l . This i s because, fo l lowing an i n i t i a l i n o c u l a t i o n , occ lus ion bodies w i l l be re leased i n the environment at each insect generat ion, thus ensuring a continuous supply of inoculum. However, sustained exposure of insects to NPVs might s e l e c t for r e s i s t a n t i n d i v i d u a l s . But in contrast to chemicals, t h i s might not be such a ser ious problem with NPVs. If the v i r u s and lepidopterans coevolve, NPVs may counter the evolut ion of host res is tance by becoming more v i r u l e n t and might s t i l l be able to contro l the pest . Hence, assessing the coevolut ionary t r a j e c t o r y of NPVs and t h e i r hosts i s c r i t i c a l i n evaluat ing the po ten t ia l of these v i ruses as long-term b i o l o g i c a l cont ro l agents. In Chapters 1 and 2, I observed that the s u s c e p t i b i l i t y of cabbage loopers (T r ichop lus ia n i ) to the singly-embedded 70 nuclear polyhedrosis v i r u s of T. n i (TnSNPV) varied among and within populations of cabbage loopers and that i t may be gen e t i c a l l y determined. In t h i s chapter, I conducted a study on the coevolution of cabbage loopers and TnSNPV. I established three l i n e s of cabbage loopers and inoculated them with v i r u s . I c o l l e c t e d the dead c a t e r p i l l a r s , i s o l a t e d the virus from the cadavers and used t h i s "selected" TnSNPV to i n f e c t the progeny of the survivors (= selected c a t e r p i l l a r s ) . At the same, I had 3 control l i n e s that were treated i d e n t i c a l l y with the exception that the larvae were mock infected with d i s t i l l e d water. If T. n i and TnSNPV coevolve, I predict that: (1) the sur v i v a l of selected c a t e r p i l l a r s treated with the o r i g i n a l v i r u s w i l l increase as the experiment progresses whereas that of controls w i l l stay the same; (2) the su r v i v a l of control c a t e r p i l l a r s fed selected TnSNPV from late i n the se l e c t i o n experiment w i l l be lower than that of control larvae inoculated with o r i g i n a l TnSNPV i . e . v i r u s c o l l e c t e d l a t e i n the se l e c t i o n experiment w i l l be more v i r u l e n t . 71 MATERIALS AND METHODS Insect and v i r u s stocks I propagated the sample of v i r u s used in Chapters 1 and 2 by feeding a small plug of d ie t contaminated with 11000 (10 u l of 1-1 x 10 OBs/ml) TnSNPV o c c l u s i o n bodies (OBs) to 100 larvae (168 hr o ld - 5th instar ) from each of the Hughes, Ignoffo, Jaques, Kaupp and Keddie populat ions (Table 1.1). Dead larvae (N = 332) were c o l l e c t e d , crushed using a mortar and p e s t l e , f i l t e r e d through 4 layers of cheesecloth and suspended in d i s t i l l e d water ( to ta l volume = 332 ml; 1 m l / l a r v a ) . The crude f i l t r a t e was centr i fuged at 700 g (2000 rpm) for 5 minutes with a GS3 S o r v a l l rotor to remove coarse t i s s u e d e b r i s . The supernatant was c o l l e c t e d and spun at 9,500 g (7,500 rpm; same rotor) for 20 minutes. The p e l l e t containing the OBs was resuspended in 200 ml of d i s t i l l e d water and washed twice. The so lu t ion was d iv ided in two samples of 100 ml and designated TnSNPVl-GO and TnSNPV2-G0. The occ lus ion body concentrat ion of each sample was determined as descr ibed in Chapter 1. I made 6 s e r i a l d i l u t i o n s of TnSNPVl-GO to 2.58 x 10 6 , 1.29 x 10 f e , 5.15 x 10 , 2.58 x 10 , 1.29 x 10 and 2.57 x 10 OBs/ml. Each d i l u t i o n was rep l i ca ted 6 t imes. I l a te r obtained a second sample of v i r u s from Dr. Jaques which was propagated by 72 feeding 14000 OBs (10 u l of 1.4 x IO** OBs/ml) to 50 larvae from of each of the 12 populat ions l i s t e d in Table 1.1. The OBs were extracted and counted in the same way and the sample designated TnSNPV3-G0. I made 5 s e r i a l d i l u t i o n s of TnSNPV3-G0 to 4.5 x l o ' ( 3.5 x 10*, 1.5 x 10*, 7.5 x 10* and 1.5 x 10 OBs/ml. Each dose was r e p l i c a t e d 3 t imes. Se lec t ion experiment Lines 1 and 2 The manipulations are summarized in Figure 3 .1 . I es tab l ished 4 (2 contro l and 2 selected) l i n e s of 500 i n d i v i d u a l s . Each of the four l i n e s consis ted of 100 neonate larvae taken from each of the Hughes, Ignoffo, Jaques, Kaupp and Keddie populat ions (Table 1.1). The larvae were reared i n d i v i d u a l l y from hatch in 25 ml p l a s t i c cups containing a h igh wheat germ d ie t (Table 1.2) at 26+1°C and 16:8 (L:D) photoperiod. At 168 hr (5th i n s t a r ) , larvae were t ransfer red to a second 25 ml cup containing only a plug of d ie t ( thickness = 2-3 mm, diameter = 5 mm). The plugs fed to larvae of se lected l i n e s 1 and 2 were t reated with 11000 OBs (10 u l of 1.1 x 10 6 OBs/ml) of TnSNPVl-GO or TnSNPV2-G0, r e s p e c t i v e l y . Plugs fed to larvae of cont ro l l i n e s 1 and 2 were t reated with d i s t i l l e d water. Larvae that consumed the 7 3 ent i re plug wi th in 24 hr were returned to t h e i r o r i g i n a l cups while those f a i l i n g to do so were discarded (approximately 3%). Larvae were checked d a i l y and diagnosis of v i r a l mor ta l i ty was based on gross pathology ( d i s c o l o r a t i o n and l i q u e f a c t i o n of the l a r v a ; Evans and Entwist le 1987). T y p i c a l l y , >_ 95% of larvae dying in se lected l i n e s exhib i ted these symptoms. None of the larvae dying i n the cont ro l l i n e s showed signs of nucleopolyhedrosis . Larvae succumbing to TnSNPV in each se lec ted l i n e were c o l l e c t e d and pooled. Occlusion bodies were i s o l a t e d from the cadavers as prev ious ly descr ibed and these se lected v i r a l samples were designated TnSNPVl-Gl ( l i n e 1) and TnSNPV2-Gl ( l i ne 2) . OB concentrat ion of both samples was determined as prev ious ly descr ibed . Samples of v i r u s were kept at -20*C while not in use. Within a given l i n e , the surv iv ing adul ts were sexed and 35-40 p a i r s mass-mated in c y l i n d r i c a l o v i p o s i t i o n cages (Chapter 1; Ignoffo 1963) wrapped with paper toweling as an o v i p o s i t i o n substrate . A b o t t l e containing 10% sucrose s o l u t i o n and a cotton wick was provided as food for a d u l t s . The towelings were c o l l e c t e d every second day and placed at 4°C to a r res t embryonic development. A f te r the four th c o l l e c t i o n , eggs were surface s t e r i l i z e d as descr ibed in 74 Chapter 1. F ive hundred neonates from each l i n e were t ransfer red to 25 ml p l a s t i c cups and used to e s t a b l i s h the next generat ion. The number of neonates taken from a given mating cage was propor t iona l to the number of females in the cage. For example, 226 males and 243 females, survived to adulthood in contro l l i n e 1 of generation 0. Thus, each of the 6 cages with 35 females contr ibuted (35 / 243) x 500 = 72 neonates to cont ro l l i n e 1 of generation 1 whereas the cage contain ing 33 females contr ibuted (33 / 243) * 500 = 68 neonates. Larvae were reared as p rev ious ly 'descr ibed and t reated with d i s t i l l e d water (control l i n e s 1 and 2) or 11000 OBs of TnSNPVl-Gl (selected l i n e 1) or TnSNPV2-Gl (se lected l i n e 2) at 168 hr (5th i n s t a r ) . Se lec t ion was c a r r i e d out fo r 8 generat ions, d iscont inued between generations 9-14, and resumed from generations 15-19. During generations 9-14, larvae were reared i n d i v i d u a l l y from hatch to adulthood in 25 ml cups but were not in fected or mock t rea ted . Line 3 At the conclusion of the experiment for l i n e s 1 and 2, I i n i t i a t e d a t h i r d cont ro l and se lected l i n e . Both l i n e s 7 5 consisted of 800 i n d i v i d u a l s and were estab l ished by taking 66 - 67 neonates from each of the 12 populat ions l i s t e d in Table 1.1. I fol lowed the same protocols as for l i n e s 1 and 2 with the exception that se lected larvae were inoculated with a marginal ly higher dose, 14000 OBs (1.4 x 10* OBs/ml) of TnSNPV3-G0, to provide a s l i g h t l y stronger s e l e c t i v e pressure . The v i r u s that was recovered from the cadavers from se lected l i n e 3 generation 0 was designated TnSNPV3-Gl and i t was used to inoculate the larvae of se lected l i n e 3 generation 1. This coevolut ion experiment was c a r r i e d out for 15 generations without i n t e r r u p t i o n . Assays of the s u s c e p t i b i l i t y of cabbage loopers to TnSNPV I monitored the evolut ion of res is tance of cabbage loopers to TnSNPV by inocu la t ing 168 hr o l d (5th ins tar ) larvae from contro l and se lected l i n e s with the i n i t i a l stocks of v i r u s . Control and se lec ted larvae were t reated i d e n t i c a l l y in these bioassays and d i f fe rences in mor ta l i ty can thus be a t t r ibu ted to p r i o r exposure of a l i n e to TnSNPV. The cadavers and surv ivors to adulthood in these assays were d iscarded. These assays are out l ined in Figure 3 .1 . 76 Lines 1 and 2 For the f i r s t s i x generations, I treated 50 la r v a e / l i n e with TnSNPVl-GO and 50 larvae/line with d i s t i l l e d water as controls. For generation 1 the dose of v i r u s was 5500 OBs (10 u l of 5.5 x 10 r OBs/ml) while a dose of 11000 OBs (10 u l of 1.1 x 10 OBs/ml) was used i n generations 2-6. For generations 7-11, I conducted multiple dose assays: larvae from each l i n e were infected with 12900, 5150, 2580, 1290 or 258 OBs (10 u l of 1.29 x 10*, 5.15 x 10^, 2.58 x 105", 1.29 x 10 , 2.58 x 10 OBs/ml, respectively) of TnSNPVl-GO. Each dose was repl i c a t e d 6 times and there were 7 larvae per r e p l i c a t e . Cohorts of 40 la r v a e / l i n e were treated with d i s t i l l e d water. For generation 12, I inoculated 75 larva e / l i n e with 25800 OBs (10 u l of 2.58 x 10 OBs/ml), a dose that had previously been shown to k i l l 100% of larvae from control l i n e s , and 25 larvae/line with d i s t i l l e d water. No l a r v a l bioassays were conducted from generations 13-19. I conducted multiple dose assays with larvae from generation 20 (offspring of the l a s t generation of selection) as described for generations 7-11. For generations 1-6 and 12, I used a T-test (2 df, 1 - t a i l probability) on ar c s i n square root transformed % sur v i v a l to detect changes i n the s u s c e p t i b i l i t y of control 77 and se lected l i n e s to TnSNPV. For generations 7-11 and 20, I ca lcu la ted the LD50 (dose required to k i l l 50% of t reated insects) with 95% confidence i n t e r v a l s for each l i n e using PROC PROBIT of SAS (1990). I compared the LD50 of i n d i v i d u a l l i n e s on the bas is of overlap of 95% confidence l i m i t s and compared the mean LD50 of contro l and se lected l i n e s by T - t e s t (2 d f , 1 - t a i l p r o b a b i l i t y ) . Line 3 At each generat ion, I t reated 50 l a r v a e / l i n e (5 r e p l i c a t e s of 10) with 7500 OBs (10 u l of 7.5 x 10^ OBs/ml) of TnSNPV3-G0 and compared the s u r v i v a l of cont ro l and se lec ted larvae with a chi -square t e s t corrected for con t inu i ty . For generations 4-16, I in fec ted an add i t iona l 30 l a r v a e / l i n e (3 r e p l i c a t e s of 10) with 35000, 15000 or 1500 OBs (10 u l of 3.5 x 10 , 1.5 x 10 or 1.5 x 10 OBs/ml, respect ive ly ) of TnSNPV3-G0 ( to ta l = 90 l a rvae ) . Cohorts of 20-25 l a r v a e / l i n e were t reated with d i s t i l l e d water at each generation as c o n t r o l s . I compared the LD50s of cont ro l and se lec ted larvae for generations 4-16 on the bas is of overlap of the 95% confidence l i m i t s ca lcu la ted by SAS. 78 Is res is tance her i tab le? To determine i f the res is tance of cabbage loopers to TnSNPV was h e r i t a b l e , 10 pa i rs of adul ts from contro l l i n e 3 generation 16 and 7 p a i r s of moths from se lected l i n e 3 generation 16 were mated in 500 ml paper cups (1 pa i r per cup; see Chapters 1 and 2) . These adul ts had not been in fected as larvae . I c o l l e c t e d the f i r s t l i n i n g of paper, s u r f a c e - s t e r i l i z e d the eggs as prev ious ly descr ibed , i n d i v i d u a l l y reared 25 larvae from each family and in fected the c a t e r p i l l a r s at 168 hr of age with 7500 OBs (10 u l of 7.5 x 10^ OBs/ml) of TnSNPV3-G0. I used the quant i ta t ive genet ic model out l ined in Chapter 2 for f u l l s i b l i n g fami l i es to estimate the h e r i t a b i l i t y of res is tance to TnSNPV. Assays of the v i ru lence of TnSNPV I monitored the evolut ion of the v i ru lence of TnSNPV by inocu la t ing cont ro l larvae with e i ther the o r i g i n a l or se lected ( i . e . v i r u s recovered from larvae se lected for res is tance) TnSNPV. Di f ferences i n s u r v i v a l of cabbage loopers in these assays can be a t t r ibu ted to change in the v i ru lence of TnSNPV. These assays are out l ined in Figure 3.1. 79 Lines 1 and 2 At generation 20, I inoculated 50 larvae from cont ro l l i n e 1 with 1000 or 2000 OBs (10 u l of 1 or 2 x 10* OBs/ml) of TnSNPVl or TnSNPV2 from generations 0 ( i n i t i a l s t o c k ) , 1, 2, 5, 7, 15, 16 or 19. I repeated the experiment with cont ro l larvae from generation 21 but t h i s time I only in fected the c a t e r p i l l a r s with 2000 OBs of TnSNPVl or TnSNPV2 from generations 0 or 19. Line 3 At generation 15, I inoculated 50 larvae of cont ro l l i n e 3 with 14000 OBs (10 u l of 1.4 x 10 6 OBs/ml) of TnSNPV3 from generations 0, 1, 2, 4, 5, 6, 10 or 15. I conducted a second assay with larvae from cont ro l l i n e 3 from generation 16 and inoculated the c a t e r p i l l a r s with 14000 OBs (10 u l of 1.4 x 10 b OBs/ml) of TnSNPV3 from generation 0, 5, 9 11 13 or 14. Analyses of v i r a l DNA using r e s t r i c t i o n enzymes I used r e s t r i c t i o n enzymes to look for genet ic changes in the v i r u s fo l lowing s e l e c t i o n . TnSNPVl-GO, TnSNPV3-G0, TnSNPVl-G20. TnSNPV2-G20 and TnSNPV3-G15 OBs were washed in 80 0.1% SDS and p e l l e t e d at 15,0003 (11000 rpm) in a Beckman JA 25.5 rotor for 30 min. The v i r i o n s were l ibe ra ted from the OBs by t rea t ing them with a l k a l i (1 M Na^COj, 150 mM NaCl , 0.01 M EDTA, pH 10.8) for 1 hr in a water bath at 37°C. The freed v i r i o n s were p e l l e t e d at 20,000a (13,000 rpm) i n a Beckman JA 25.5 rotor for 1 hr , resuspended in proteinase-K buf fer (10 mM T r i s , 10 mM EDTA, 150 mM NaCl , 0.4% SDS, pH 7.4) and digested with prote inase-K (200 ug /u l ) overnight at 37°C. DNA was p u r i f i e d by sequent ia l ext ract ions i n phenol , phenol:chloroform:isoamyl a lcohol (25:24:1) and chloroform:isoamyl a lcohol (24:1). DNA was p r e c i p i t a t e d from the f i n a l aqueous phase by adding 1/10 volume of 2 M sodium acetate (pH = 5.2) and 2 volumes of 100% ethanol and s tor ing at - 20°C for 2 hr . The DNA from each sample was p e l l e t e d at 2800g (3500 rpm) in a Beckman GS-6R centr i fuge for 1-1.5 h r , washed with 70% and 100% ethanol , d r ied at 55°C for 5 min and resuspended in s t e r i l e d i s t i l l e d water. P u r i f i e d DNA (2 ug) from each sample was digested with BamI, EcoRI, H ind i I I , Ps t I , S a i l or Sst I i n the appropriate buf fer system for 2 hr at 37*C. The DNA r e s t r i c t i o n endonuclease fragments were separated by e lec t rophores is on 0.7% agarose hor izonta l submarine gel i n a buf fer containing 0.5 ug/ml of ethidium bromide at 45 V overnight . Lambda DNA cut with HindiI I was analyzed concurrent ly as a molecular weight marker. 81 RESULTS Surv iva l of l i n e s during s e l e c t i o n Lines 1 and 2 During s e l e c t i o n , the mean % s u r v i v a l of cont ro l l i n e s 1 and 2 was always be t te r , and on average 2.6 times greater , than that of se lec ted l i n e s 1 and 2 (Figure 3.2A). The mean % s u r v i v a l of se lected l i n e s 1 and 2 improved during the per iod of s e l e c t i o n (Pearson r = 0.696, P = 0.006, N = 14) whereas there was a s l i g h t but not s i g n i f i c a n t dec l ine in the mean % s u r v i v a l of cont ro l l i n e s 1 and 2 (Pearson r = -0.374, P = 0.188, N = 14; Figure 3.2A). The mean % s u r v i v a l of the cont ro ls was l e s s than 92% only i n generat ion 17 (72%; Figure 3.2A). In that generat ion, I d iscarded many contro l c a t e r p i l l a r s because there was an outbreak of fungus on the d i e t and I wanted to prevent the fungus from spreading sys temica l l y . When s e l e c t i o n was discont inued (generations 9-14), the mean % s u r v i v a l of contro l and se lec ted l i n e s 1 and 2 were near ly i d e n t i c a l (Figure 3.2A). Line 3 Results from cont ro l and se lected l i n e s 3 were 82 q u a l i t a t i v e l y the same as that of l i n e s 1 and 2. The % s u r v i v a l of contro l l i n e 3 was on average 2.7 times bet ter than that of se lec ted l i n e 3 (Figure 3.2B). The % s u r v i v a l of contro l l i n e 3 improved s l i g h t l y but not s i g n i f i c a n t l y (Pearson r = 0.424, P = 0.102, N = 16), whereas the % s u r v i v a l of se lected l i n e 3 improved s i g n i f i c a n t l y over the same per iod (Pearson r = 0.747, P < 0.001, N = 16). Assays of the s u s c e p t i b i l i t y of cabbage loopers to TnSNPV Lines 1 and 2 In the f i r s t generat ion, the assay sample of c a t e r p i l l a r s from cont ro l and se lected l i n e s 1 and 2 d id not d i f f e r s i g n i f i c a n t l y in t h e i r s u r v i v a l when chal lenged with 5500 OBs of TnSNPVl-GO (mean + SE; contro l l i n e s : 35 + 2%; se lected l i n e s ; 14 ± 10%; t = 1.726, P =. 0.113). The poor mean % s u r v i v a l of the se lec ted l i n e s was due to only 3/50 c a t e r p i l l a r s from se lected l i n e 1 surv iv ing i n f e c t i o n . The mean % s u r v i v a l of se lec ted l i n e 1 and 2 was higher than that of cont ro l l i n e s 1 and 2 in generations 2 and 4 - 6 (Figure 3.3A) and the d i f fe rence was s i g n i f i c a n t in generation 2 (t = -2.712, P = 0.055) and generation 5 (t = - 3.999, P = 0.029). 83 For generations 7 to 9 r e s p e c t i v e l y , the mean LD50 of se lected l i n e s 1 and 2 was 1.9, 2.8 and 3.3 times bigger than that of cont ro l l i n e s 1 and 2, and the d i f fe rence was s i g n i f i c a n t at generation 9 (Figure 3.3B; generation 7: t = - 1.456, P = 0.142; generation 8: t = -1 .269, P = 0.166; generation 9: t = -6 .543, P = 0.012). Moreover, at generation 9, the LD50 of se lec ted l i n e s 1 and 2 were both greater than those of e i ther contro l l i n e s 1 or 2 based on overlap of 95% c . i . (Table 3 .1) . At generation 10, the LD50s of cont ro l and se lec ted l i n e s increased even though s e l e c t i o n had been stopped in generation 9. Nevertheless, the mean LD50 of the se lec ted l i n e s was 3.5 times greater than that of the cont ro ls (s imi la r to generation 9) and the d i f fe rence was s i g n i f i c a n t (Figure 3.3B; t = -3.774, P = 0.032). Apart for a s l i g h t overlap i n the 95% c . i . of contro l l i n e 2 and se lected l i n e 2, the LD50 of se lected l i n e s 1 and 2 were greater than those of cont ro l l i n e s 1 and 2 (Table 3 .1) . The LD50s of a l l l i n e s in generation 11 were comparable to those in generation 9. The mean LD50 of se lec ted l i n e s was 2.9 times greater than contro ls (Figure 3.3B; t = -3 .733, P = 0.033). The 95% c . i . of se lected l i n e 2 overlapped s l i g h t l y with those of contro l l i n e 2 but otherwise the LD50s 84 of se lec ted l i n e s 1 and 2 were greater than those of cont ro l l i n e s 1 and 2 (Table 3 .1) . In generation 12, I conducted a s i n g l e dose assay and inoculated larvae with 25800 TnSNPVl-GO OBs. Only 2% (SE = 1%) of larvae from contro l l i n e s 1 and 2 survived t h i s dose compared to 27% (SE = 1%) for c a t e r p i l l a r s from se lected l i n e s 1 and 2 (t = - 10.523, P = 0.005). At generat ion 20 (= o f f s p r i n g of the l a s t generat ion of s e l e c t i o n ) , the mean LD50 of se lected l i n e s 1 and 2 was 4.4 times greater than that of contro l l i n e s 1 and 2 (Figure 3.3B; t = -14.81, P = 0.003). The LD50 of se lected l i n e 2 was greater than that of cont ro l l i n e s 1 and 2 (Table 3 .1) . The LD50 of se lected l i n e 1 was a lso considerably larger than that of both cont ro l l i n e s but SAS was unable to compute 95% c . i . presumably because mor ta l i ty d id not d i f f e r much across the f i v e TnSNPV doses used i n that assay (Table 3 .1 ) . Line 3 There was a s l i g h t but nons ign i f i can t dec l ine in the surv iva l of contro l larvae inoculated with 7500 OBs of TnSNPV3-G0 from generations 1 to 15 (Figure 3.4A; Pearson r = -0.247, P = 0.23, N = 15). Conversely, the 85 s u r v i v a l of se lec ted larvae t reated with the same dose improved s i g n i f i c a n t l y over that per iod (Figure 3.4A; Pearson r = 0.627, P = 0.015, N = 15). From generations 7 to 15, the s u r v i v a l of larvae from se lected l i n e 3 was s i g n i f i c a n t l y greater than that of c a t e r p i l l a r s from cont ro l l i n e 3 ( A l l Yates' chi -square > 5.63 and P < 0.025). The LD50 of se lec ted l i n e 3 increased from generation 4 to 15 (Pearson r = 0.269, P = 0.200; Spearman r = 0.804, P < 0.001, N = 12) but not that of cont ro l l i n e 3 (Pearson r = 0.010, Spearman r = 0.091, both P 's > 0.75, N = 15). The LD50 of se lec ted l i n e 3 d i f f e r e d (based on overlap of 95% c . i . ) from that of cont ro l l i n e 3 at generations 7-10, 14 and 15 (Figure 3.4B). In a d d i t i o n , the LD50 of se lected l i n e 3 for generations 11 and 13 were considerably greater than cont ro l l i n e 3 but SAS was unable to ca lcu la te confidence in te rva ls presumably because s u r v i v a l d id not vary much and was greater than 73% for a l l doses. At generation 15, the LD50 of se lected 3 (60851 OBs) was 25 times greater than that of cont ro l l i n e 3.(2406 OBs; Figure 3.4B) . 86 Is res is tance her i tab le ? At generation 16, the s u r v i v a l fo l lowing chal lenge with v i r u s of o f f s p r i n g from se lected l i n e 3 pa i rs of moths (mean + SE: 61 + 6%, N = 7) was s i g n i f i c a n t l y higher than that of progeny from cont ro l l i n e 3 parents (mean + SE: 12 + 3%, N = 10; t = -7 .97 , df = 15, P < 0.001). This y ie lded a h e r i t a b i l i t y for res is tance to TnSNPV of 0.96 (P < 0.001). Assays of the v i ru lence of TnSNPV Lines 1 and 2 The s u r v i v a l of cont ro l larvae ( l i n e 1 generation 20) improved, a l b e i t not s i g n i f i c a n t l y , with the generation from which the v i r u s was i s o l a t e d (Figure 3.5; 1000 OBs: Pearson r = 0.612, P = 0.107, N = 8; 2000 OBs: Pearson r = 0.614, P = 0.106, N = 8) . However, these re la t ionsh ips appear to be caused by the high s u r v i v a l of i n d i v i d u a l s in fected with v i r u s from generation 16 (nearly 100%). When I repeated the ana lys is without the data from generation 16, the r e l a t i o n s h i p was much weaker (1000 OBs: Pearson r = 0.509, P = 0.243, N = 7 ; 2000 OBs: Pearson r = 0.577, P = 0.175, N = 7) . The mean s u r v i v a l of contro l larvae ( l ine 1 generation 21) inoculated with 2000 87 OBs of o r i g i n a l v i r u s (mean + SE: 53 + 13%) d id not d i f f e r from those treated with v i r u s from generation 19 (mean +_ SE: 64 + 1%; t = - 0.917, P = 0.456, df = 2) . Line 3 In both t r i a l s for the assay of the v i ru lence of TnSNPV3, the s u r v i v a l of ind iv idua ls from contro l l i n e 3 was lower when they were in fec ted with v i r u s extracted la te in the s e l e c t i o n experiment i . e . la te v i r u s appeared more v i r u l e n t (Figures 3.6A-B, T r i a l 1; Pearson r = -0 .298, P = 0.236, N = 8; T r i a l 2; Pearson r = -0 .373, P = 0.233, N = 6) . The s u r v i v a l of contro l larvae t reated with TnSNPV3-G0 was higher in T r i a l 2 (Yates 1 chi -square = 6.83, P < 0.01) but there was no d i f fe rence between t r i a l s for c a t e r p i l l a r s in fected with TnSNPV3 from generation 5 (Figure 3.6B; Yates 1 ch i -square = 0.761, 0.25 < P < 0.10). Nevertheless, I pooled the two t r i a l s and used the mean s u r v i v a l for generations 0 and 5. The s u r v i v a l of cont ro l i n d iv idua ls decreased with the generation from which the v i r u s was extracted but the r e l a t i o n s h i p was again not s i g n i f i c a n t (Figure 3.6C, Pearson r = -0.404, P = 0.097, N = 12). I f , for some reason, the c a t e r p i l l a r s used i n the second assay were less suscept ib le than those in the f i r s t assay, then the s u r v i v a l of i n d i v i d u a l s in assay 2 should be 88 corrected and reduced. This would fur ther exacerbate the d i f fe rence between the v i ru lence of TnSNPV3 i s o l a t e s c o l l e c t e d ear ly and la te in the s e l e c t i o n experiment. Analyses of v i r a l DNA using r e s t r i c t i o n enzymes The r e s t r i c t i o n endonuclease fragment p r o f i l e s of the DNAs of the 5 TnSNPV i s o l a t e s were i d e n t i c a l i n a l l but three cases (Figures 3 .7A-F) . On each of these occas ions , i t was the p r o f i l e of TnSNPVl generation 19 that d i f f e r e d from the others . One fragment was missing from each of the r e s t r i c t i o n p r o f i l e s of TnSNPVl generation 19 digested with BamHI, H i n d l l l and SstI (Figures 3.7A, C and F ) . 89 DISCUSSION Se lec t ion should favour c a t e r p i l l a r s that are r e s i s t a n t to nuclear polyhedrosis v i r u s e s . Conversely, t ransmission of the disease i s dependent upon death of the host and s e l e c t i o n should favour NPV genotypes that are s u f f i c i e n t l y v i r u l e n t to overcome host defences. The coevolut ion of c a t e r p i l l a r s and NPVs might thus lead to an arms-race. Evolut ion of res is tance of cabbage loopers to TnSNPV The s u r v i v a l of T. n i inoculated with coevolved TnSNPV improved through time i n a l l three l i n e s (Figure 3 .2) . The improved s u r v i v a l of c a t e r p i l l a r s was due to s e l e c t i o n for larvae res is tan t to the d isease . Af ter 14 generations of s e l e c t i o n , the mean LD50 of se lected l i n e s 1 and 2 was 4.4 times greater than that of cont ro l l i n e s 1 and 2. A f te r 15 generations of s e l e c t i o n , the LD50 of se lec ted l i n e 3 was 25 times larger than that of contro l l i n e 3. Three other s tudies have reported s e l e c t i o n for res is tance to bacu lov i ruses . The LD50 of Phthorimaea o p e r c u l e l l a increased 6 f o l d a f te r s e l e c t i o n with a granulos is v i r u s for 10 generations (Br iese and Mende 1983). The LD50 of Spodoptera f rugiperda was 4.5 greater a f te r 90 exposure to a NPV for 7 generations (Fuxa et a l . 1988). Fol lowing exposure to a granulos is v i r u s fo r two years , the LD50 of P l o d i a i n t e r p u c t e l l a se lected for res is tance was twice as high as that of cont ro ls (Boots and Begon 1995). There are a lso c i rcumstant ia l reports of res is tance to NPVs or granulosis v i r u s e s . For example, the LD50 of a populat ion of l a rch budmoths (Eucosma griseana) fo r a granulos is v i r u s was 38 times greater a f te r an outbreak of the disease in the f i e l d (Martignoni 1957). Two explanations may be given to account for the greater res is tance achieved in l i n e 3 compared to l i n e s 1 and 2. F i r s t , s e l e c t i o n may have been marginal ly s t ronger . Larvae from se lected l i n e 3 were fed 14000 OBs at each generation whereas c a t e r p i l l a r s in se lec ted l i n e s 1 and 2 were inoculated with only 11000 OBs. Second, se lected l i n e 3 may have been more g e n e t i c a l l y va r iab le and more l i k e l y to include a rare gene coding for r e s i s t a n c e . Selected l i n e 3 consis ted of 800 i n d i v i d u a l s and i t was es tab l ished by taking neonates from 12 T. ni. populat ions. In cont ras t , se lected l i n e s 1 and 2 consisted of only 500 i n d i v i d u a l s taken from 5 T. ni. populat ions (see Mater ia ls and Methods). In a l l three l i n e s , s e l e c t i o n required 7-9 generations to s i g n i f i c a n t l y a f fec t the s u s c e p t i b i l i t y of T. ni. to 91 TnSNPV. However, res is tance could have evolved sooner and gone undetected. Although comparing LD50s i s adequate for documenting res is tance at high f requencies , i t i s not very s e n s i t i v e to small changes in the frequency of res is tance (Roush and M i l l e r 1986, f f rench-Constant and Roush 1990). Comparison of s u r v i v a l at doses that k i l l 99.9% or more of suscept ib le i n d i v i d u a l s i s more e f f i c i e n t at detect ing res is tance when i t f i r s t appears in a populat ion ( f f rench-Constant and Roush 1990). Resistance to pathogens and chemicals f requent ly involves f i t n e s s costs and i s unstable . If r e s i s t a n t in d iv idua ls have poorer reproductive success than uninfected suscept ib le i n s e c t s , then r e s i s t a n t l i n e s can revert to s u s c e p t i b i l i t y when the s e l e c t i v e pressure i s removed (e .g . Fuxa and Richter 1989; Bush et a l . 1993, Cochran 1993, Tabashnik et a l . 1994). S p e c i f i c a l l y , Fuxa and Richter (1989) showed that S. f rugiperda which were r e s i s t a n t to NPV reverted to s u s c e p t i b i l i t y wi th in 1 generation when s e l e c t i o n with NPV was d iscont inued. When I d iscont inued s e l e c t i o n , the res is tance of T. n i decayed only very s l i g h t l y . Two generations a f te r having stopped s e l e c t i o n , the res is tance r a t i o (mean LD50 of cont ro l l i n e s 1 and 2 / mean LD50 of se lec ted l i n e s 1 and 2) only dec l ined from 3.3 (generation 9) to 2.9 (generation 11). Moreover, the s u r v i v a l of larvae from 92 selected l i n e s 1 and 2 was s t i l l considerably higher than that of contro l l i n e s 1 and 2 at generation 12 i . e . 3 generations a f te r having discont inued s e l e c t i o n . Although these f ind ings suggest that the res is tance of cabbage loopers to TnSNPV may be s t a b l e , I cannot conclude that i t does not incur any f i t n e s s c o s t s . That i s because i f most of the suscept ib le ind iv idua ls had been removed, and se lec ted l i n e s 1 and 2 were composed predominantly of r e s i s t a n t i n d i v i d u a l s at generation 9 (when s e l e c t i o n was in te r rupted) , revers ion may not occur or may have required more time to occur . In a d d i t i o n , res is tance to TnSNPV may e n t a i l costs other than those d i r e c t l y re la ted to f i t n e s s . For example, Fuxa and Richter (1990) showed that S. f rugiperda r e s i s t a n t to an NPV were more suscept ib le to the chemical i n s e c t i c i d e methyl parath ion. I w i l l formal ly address the issue of whether the res is tance of cabbage loopers to TnSNPV i s c o s t l y in Chapter 4. Mechanisms of res is tance of T. n i to TnSNPV A va r i e ty of mechanisms may expla in the increased res is tance of se lected l i n e s to TnSNPV. I address these poss ib le mechanisms as the i n f e c t i o n progresses through the i n s e c t . F i r s t , once in the midgut, the occ lus ion body matrix i s d isso lved and the v i r i o n s are re leased . The d i s s o l u t i o n of 9 3 the matrix occurs opt imal ly under high a l k a l i n e condi t ions (e.g P r i t c h i t t et a l . 1984). Insects with suboptimal pH, in terms of OB d i s s o l u t i o n , might thus be l ess suscept ib le to the disease (see Keating et a l . 1989). Second, the re leased v i r i o n s are very suscept ib le to i n a c t i v a t i o n by the d iges t ive f l u i d s of the midgut ( e .g . Elam et a l . 1990) and must r a p i d l y pass through the p e r i t r o p h i c membrane to reach the midgut epithel ium where they can i n i t i a t e an i n f e c t i o n (Granados and Lawler 1981). NPVs may have a prote in that can d isrupt the p e r i t r o p h i c membrane thus al lowing f ree passage of the v i r i o n s (Derksen and Granados 1988). Larvae that possess p e r i t r o p h i c membranes that can withstand d i s r u p t i o n and t rap v i r i o n s may be l ess suscept ib le to NPVs (but see Begon et a l . 1993, Engelhard and Volkman 1995). Larvae with greater concentrat ion of gut proteases could be less suscep t ib le . The d iges t ive f l u i d s of B. mori have been suggested to contain a n t i v i r a l substances which can agglut inate NPV v i r i o n s (e .g . Aizawa 1962, Mukai et a l . 1969, Aratake and Ueno 1973, Uchida et a l . 1984, Funakoshi and Aizawa 1989). Larvae with higher concentrat ions of such a n t i v i r a l substances may be less suscept ib le to NPVs. T h i r d , the v i r i o n s enter midgut c e l l s by a mechanism that i s not f u l l y understood but i s l i k e l y to involve the 94 binding of the v i r i o n s to receptors on the surface of c e l l s . Larvae lack ing these putat ive c e l l receptors may be less suscept ib le to the d isease . Keddie et a l . (1989) showed that in fected midgut columnar c e l l s are sloughed into the lumen of the midgut. Larvae that slough o f f in fec ted c e l l s f as te r than the v i r u s r e p l i c a t e s could be less suscept ib le to NPVs. Fourth, v i r i o n s produced i n the midgut penetrate the hemocoel and spread the disease sys temica l l y . Hemocytes o f f e r one poss ib le l i n e of defense in the hemocoel. Some hemocytes are capable of phagocytosing fore ign p a r t i c l e s such as v i ruses (Wigglesworth 1972). Begon et a l . (1993) showed that hemocytes appear to consume granulosis v i r u s p a r t i c l e s f l o a t i n g i n the hemolymph and to take them to the Malpighian tubules for excret ion (see a lso Hess and Falcon 1987). F i f t h , pupation appears to d is rupt NPV r e p l i c a t i o n (Murray et a l . 1991 and references t h e r e i n ) . The eqt gene of NPVs inac t iva tes the moulting hormones of c a t e r p i l l a r s thus prolonging the l a r v a l stage ( O ' R e i l l e y and M i l l e r 1991, O ' R e i l l e y 1995). Insects that are not suscept ib le to the e f f e c t of eqt may thus be able to escape and pupate before the v i r u s can k i l l them. To date, the evidence suggests that res is tance to NPVs 95 involves mainly defense mechanisms i n the gut (mechanisms 1-3 above; Br iese 1981). Fuxa and Richter (1989) showed that S. f rugiperda se lected for res is tance were equal ly suscept ib le as cont ro ls when v i r i o n s were in jec ted d i r e c t l y into the hemocoel thus bypassing the midgut. Evolut ion of the v i ru lence of TnSNPV The evolut ion of res is tance in T. ni. was not accompanied by a s t a t i s t i c a l l y s i g n i f i c a n t change i n the v i ru lence of TnSNPV in any of the three l i n e s . However, some evidence suggests that TnSNPV3 may have evolved higher v i r u l e n c e : (1) i n the assays for v i r u l e n c e , the s u r v i v a l of c a t e r p i l l a r s from cont ro l l i n e 3 dec l ined with the generat ion from which the v i r u s was i s o l a t e d ; (2) i n sp i te of larvae from se lected l i n e 3 showing 25 f o l d res is tance , 14000 OBs of TnSNPV3 from generation 15 was s t i l l capable of k i l l i n g about 50% of se lected larvae at the conclusion of the s e l e c t i o n experiment. Se lec t ion may have favoured increased v i ru lence in l i n e 3 because larvae evolved considerably higher res is tance than in l i n e s and 1 and 2 (4.4 f o l d ) . This would r e - a s s e r t that the evolut ion of the v i r u s i s t i g h t l y determined by the evolut ion of the host (Janzen 1980). The v i ru lence of T. n i mutiply-embedded nuclear polyhedrosis and Mammestra brass icae multiply-embedded nuclear polyhedrosis 96 v i r u s d i d not change when passed i n cabbage loopers for 15 and 25 generat ions, respec t ive ly (Potter et a l . 1978, C r o i z i e r et a l . 1985). However, in those experiments, larvae were not concurrent ly se lected for r e s i s t a n c e . Apart for one fragment missing in each of the r e s t r i c t i o n endonuclease p r o f i l e s of the DNA of TnSNPVl from generation 19 digested with BamHI, H i n d l l l and S s t I , the p r o f i l e s of a l l TnSNPV i s o l a t e s were i d e n t i c a l . However, these genet ic analyses are f a i r l y crude and mutations outs ide the r e s t r i c t i o n s i t e s (other than large de le t ions or inser t ions ) w i l l not be detected. This i s important as one or a few point mutations can a f fec t the v i ru lence of v i ruses ( e . g . p o l i o v i r u s , Westrop et a l . 1989; in f luenza v i r u s , Webster et a l . 1986). The Oryctes v i r u s ( u n t i l recent ly a lso a member of the Baculovir idae) has been released to contro l the coconut palm rhinoceros beet le (Oryctes rhinoceros) on small a t o l l s in the South P a c i f i c (Marshall 1970, Zelazny et a l . 1990). Crawford and Zelazny (1990) monitored the evolut ion of the v i r u s on some of these is lands using r e s t r i c t i o n enzymes and observed only three genomic changes in the four years s ince the v i r u s had been released (= an estimated 54,000 cyc les of r e p l i c a t i o n ) . However, Crawford and Zelazny (1990) d id not compare the v i ru lence of those v i r a l i s o l a t e s . 97 Three reasons can be given to expla in why the v i r u s may not have evolved increased v i r u l e n c e . F i r s t , there may have been l i t t l e v a r i a t i o n i n the o r i g i n a l v i r a l samples. Although the two TnSNPV samples obtained from Dr. Jaques were not c l o n a l i s o l a t e s and were i s o l a t e d from insec ts on d i f f e r e n t occas ions, t h e i r r e s t r i c t i o n p r o f i l e s were i d e n t i c a l . Moreover, the r e s t r i c t i o n p r o f i l e of the o r i g i n a l TnSNPV digested with Ecor l was i d e n t i c a l to that of Smith and Summers (1982). Th is suggests that the TnSNPV genome may be very s tab le and that there might be l i t t l e genet ic v a r i a t i o n wi th in the TnSNPV spec ies . Second, low mutation rates may have constrained the evolut ion of the v i r u s . The res is tance of larvae to TnSNPV may only involve the loss of a receptor s i t e fo r the v i r u s and could occur v i a a point mutation, d e l e t i o n or i n s e r t i o n (Lenski and Levin 1985). Conversely, increased v i ru lence may require the synthesis of an e n t i r e l y novel p ro te in and more extensive genet ic modi f icat ions i . e . i t may be simpler for c a t e r p i l l a r s to lose a funct ion than for the v i r u s to gain one. T h i r d , the 25 - fo ld increase i n res is tance may not have been a s u f f i c i e n t l y strong s e l e c t i v e pressure on the v i r u s to favor the evolut ion of increased v i r u l e n c e . 98 Poss ib le sources of er ror and e f f e c t on r e s u l t s The s ing le most important source of er ror in t h i s study concerns the counting of occ lus ion bodies . Although I used the mean of 8 - 10 hemacytometer counts to determine the OB concentrat ion of each TnSNPV sample, the counts were qui te v a r i a b l e . On some occasions there was a 2 f o l d d i f fe rence between the lowest and highest count. Thus, during the s e l e c t i o n experiment, larvae may have been in fected with a lower or higher dose than intended ( i . e . 11000 or 14000 OBs). This may p a r t i a l l y expla in the jagged s u r v i v a l of the se lec ted l i n e s in the s e l e c t i o n experiment (Figure 3 .2) . I t could a lso expla in the o u t l i e r points in the the assays of the v i ru lence of TnSNPV i s o l a t e s (Figures 3.5 and 3 .6) . However, in the assays of the v i ru lence of TnSNPV3, i t i s somewhat u n l i k e l y that the poor s u r v i v a l of cabbage loopers in fected with TnSNPV3 from generation 9 , 10, 13, 14 and 15 a l l resu l ted from larvae rece iv ing considerably more OBs than c a t e r p i l l a r s t reated with v i r u s from e a r l i e r generat ions. Er rors in counting occ lus ion bodies w i l l not a f fec t the assays of l a r v a l res is tance s ince the o r i g i n a l v i r a l stocks were s e r i a l l y d i l u t e d at the beginning of both s e l e c t i o n experiments and the same d i l u t i o n s were used to i n f e c t cont ro l and se lected larvae at each generat ion. 99 A second poss ib le source of er ror concerns the age of larvae at time of i n f e c t i o n . Engelhard and Volkman (1995) showed that small d i f fe rences (6 hr) i n the age of c a t e r p i l l a r s could a f fec t t h e i r s u s c e p t i b i l i t y to NPVs. During s e l e c t i o n and i n a l l assays ( for res is tance and v i ru lence) larvae were inoculated at 168+.1 hr a f te r having been placed on food. Thus, wi thin a generat ion, contro l and se lected larvae should have been at s i m i l a r p h y s i o l o g i c a l ages. T h i r d , i t i s genera l ly be l ieved that the v i ru lence of NPVs i s not a f fected by storage time at -20 C. However, i f the v i ru lence of TnSNPV i s o l a t e s d id decay with storage t ime, then the o r i g i n a l TnSNPV would be less v i r u l e n t than TnSNPV recovered from the l a s t generation of the s e l e c t i o n experiment. This would obviously a f fec t the r e l i a b i l i t y of the assays for the v i ru lence of TnSNPV. However, t h i s d id not appear to happen. In the assays for cabbage looper r e s i s t a n c e , the LD50 of the o r i g i n a l TnSNPVl and TnSNPV3 for contro l c a t e r p i l l a r s d id not increase as the experiment progressed (Figures 3.5 and 3.6) . Th is suggests that storage time had minimal e f f e c t s on the v i ru lence of TnSNPV i s o l a t e s . Conclusions and Impl icat ions for B i o l o g i c a l Control The r e s u l t s of t h i s study do not support the Red Queen 100 hypothesis (Van Valen 1973). Cabbage loopers evolved res is tance to TnSNPV but the v i r u s d id not keep pace by becoming s t a t i s t i c a l l y more v i r u l e n t . TnSNPV3 i s the only v i r u s that may have evolved increased v i r u l e n c e . However, regardless of whether TnSNPV d i d evolve increased v i r u l e n c e , cabbage loopers seem to have taken the lead in the arms-race. The s u r v i v a l of T. n i in fec ted with the pu ta t i ve ly coevolving TnSNPV increased and was higher at the conclusion of both s e l e c t i o n experiments. Although cabbage loopers evolved up to 25 - fo ld res is tance to TnSNPV and the v i rus d id not become more v i r u l e n t , i t might s t i l l be poss ib le to use TnSNPV as a f a i r l y successfu l long-term cont ro l agent against cabbage loopers . At the conclusion of the s e l e c t i o n experiment, 11000 OBs of TnSNPVl/TnSNPV2 from generation 19 and 14000 OBs of TnSNPV3 from generation 15 were s t i l l able to k i l l about 50% of se lec ted larvae and t h i s may provide adequate crop p r o t e c t i o n . Zelazny et a l . (1989, 1990) showed that even though the coconut palm rhinoceros beet le may have evolved res is tance to the Oryctes v i r u s , the beet le remained at low l e v e l s and damage to palm trees was less on a t o l l s where the v i r u s had been re leased 4 years e a r l i e r than on cont ro l i s l a n d s . See B i rd (1961), Smirnoff et a l . (1962) and Otvos et a l . (1993) for other examples of prolonged cont ro l of insects fo l lowing a s ing le in t roduct ion of NPVs. 101 CM T3 CD +—> o _CD CD co CM TJ- CO CD Is-CO if) o CM CJ) CD CD cn CD CD CO ID CO 00 in CO CD . o 00 oo CO o CM * 5 CO "1 CD LO "--m CD CD IS-| s . CD m ,_" CM o CO in IS-CM CO CO in Is-T J CD -4—' o _CD CD CO m 1—1 oo CM 00 o O Is- 00 o o in CO CM 00 CO CO cn 00 o" cn r-" CM" Is- 00 co CM CO 00 7~. 7~. CM CD O CD in o h-CD "R~_ co oo CM in CO CO cn CM CD IS-00 00 o o" o IS-co o o oo CD o CM T J c CO CM "o c o o CO o m in in co" IS-cn cn CM m CM TI-" cn o CD „ in co co 2 3 Is-cn 00 in CM CD CO CD CM CD cn CD IS- cn CO CO I — CD cn CM r-T CM" CM CM oo CM in o CO 7~. 7~. w c o CO CD c CD CJ) CO c o O Is- 1 1 CD O) o CO Is- i — ^ — CO 1 — CO CM CO CM CM co CM Is- cn CO CM" CO CM" CD 00 o O I — 7~. T— . 7~. o m CD oo o CD CO CM Is-co co ™ CD CM 2 o" i — cn in 00 CM Is- CJ) CM c o "•4—» CO CD c CD es 00 O) o CM 102 Figure 3 .1 . Flowchart summary of s e l e c t i o n experiments and assays used to detect changes i n res is tance and v i r u l e n c e . Control and se lec ted l i n e s 1 and 2 were es tab l ished by taking 100 larvae from each of the HU, I, J , KA, and KE populat ions (N = 500 c a t e r p i l l a r s per l i n e ) ; cont ro l and se lec ted l i n e s 3 were estab l ished by taking 66 - 67 larvae from the AB, AZ, FE , FO, HA, HU, I, J , KA, KE, L and S populat ions (N = 800 c a t e r p i l l a r s per l i n e ) . At 168-hr-of-age (5th i n s t a r ) , larvae from se lected l i n e s 1 and 2 were in fec ted with 11000 OBs of TnSNPVl-GO or TnSNPV2-G0, r e s p e c t i v e l y , and c a t e r p i l l a r s from se lected l i n e 3 with 14000 OBs of TnSNPV3-G0. Within a given l i n e , the surv ivors were mass-mated and the cadavers were c o l l e c t e d , pooled and the v i r u s extracted from the corpses. Th is "selected" TnSNPV (TnSNPVl-Gl, TnSNPV2-Gl or TnSNPV3-Gl) was used to inoculate the next generation of insec ts (N = 500 fo r se lec ted l i n e s 1 and 2; N = 800 for se lected l i n e 3). Cabbage loopers in contro l l i n e s were t reated i d e n t i c a l l y with the exception that 168-hr-o ld larvae were mock-infected with d i s t i l l e d water. The v i rus recovered from the i n d i v i d u a l s succumbing to TnSNPV i n generation 1 (TnSNPVl-G2, TnSNPV2-G2 or TnSNPV3-G2) was used to i n f e c t generation 2 c a t e r p i l l a r s of se lected l i n e s 1, 2 or 3, r e s p e c t i v e l y . For l i n e s 1 and 2, t h i s s e l e c t i o n treatment was conducted for generations 0-8, d iscont inued between generations 9-14 and resumed from generations 15-19. For l i n e s 3, s e l e c t i o n was conducted for 15 generations without i n t e r r u p t i o n s . LARVAL BIOASSAY: The evolut ion of cabbage looper res is tance to TnSNPV was monitored by inocu la t ing 168-hr-old contro l and se lected larvae with the o r i g i n a l TnSNPV (11000 OBs of TnSNPVl-GO for cont ro l and se lec ted l i n e s 1 and 2; 14000 OBs of TnSNPV3-G0 for cont ro l and se lec ted l i n e s 3) . In these assays, d i f fe rences in the s u r v i v a l of cabbage loopers can be a t t r ibu ted to p r i o r exposure of a l i n e to TnSNPV. VIRAL BIOASSAY: The evolut ion of TnSNPVl and TnSNPV2 v i ru lence was monitored by inocu la t ing 168-hr o ld larvae from cont ro l l i n e 1 with e i ther the o r i g i n a l or se lected TnSNPVl or TnSNPV2. S i m i l a r l y , the evolut ion of the v i ru lence of TnSNPV3 was monitored by inocu la t ing c a t e r p i l l a r s from contro l l i n e 3 with e i ther the o r i g i n a l or se lec ted TnSNPV3. In these assays, d i f fe rences in the s u r v i v a l of larvae can be a t t r ibu ted to changes i n the v i ru lence of TnSNPV. 103 104 Figure 3.2 Percent s u r v i v a l of l i n e s during s e l e c t i o n . (A) Mean (+1 SE) percent s u r v i v a l of control (•) and selected (•) l i n e s 1 and 2. There was no selection between generations 9 and 14. (B) Percent survival of control (•) and selected (•) l i n e 3. 105 1 0 0 (B) Line 3 0 1 0 1 5 Generation 106 Figure 3.3 (A) Mean (+1 SE) percent s u r v i v a l of larvae from cont ro l (A) and se lec ted (•) l i n e s 1 and 2 inoculated with 11000 OBs of TnSNPVl-GO for generations 2 to 6. (B) Mean (+1 SE) LD50 of contro l (A) and selected (•) l i nes 1 and 2 t reated with 12900, 5150, 2580, 1290 or 258 OBs of TnSNPVl-GO. 107 2 5 0 0 0 2 0 0 0 0 1 5 0 0 0 1 0 0 0 0 5 0 0 0 0 (B) T i i i i r 8 1 0 1 2 1 4 1 6 1 8 2 0 Generation 108 Figure 3 .4 (A) Surv iva l of larvae from cont ro l (•) and se lected (•) l i n e 3 inoculated with 7500 OBs of TnSNPV3-G0 for generations 1-15. (B) LD50 and 95% confidence i n t e r v a l s of cont ro l (A) and se lec ted (•) l i n e 3 t reated with 35000, 15000, 7500 or 1500 OBs of TnSNPV3-G0• 109 (A) 1 0 0 i T 1 1 1 1 < 1 1 1 1 r 2 4 6 8 1 0 1 2 1 4 2 0 0 0 0 0 0 4 8 0 0 0 0 1 9 0 0 0 0 6 0 0 0 0 4 0 0 0 0 2 0 0 0 0 0 8 1 0 1 2 1 4 Generation 110 Figure 3.5 Mean (+1 SE) percent s u r v i v a l of larvae from cont ro l l i n e 1 t reated with 1000 (•) or 2000 OBs (•) of TnSNPVl or TnSNPV2 from generations 0, 1, 2, 5, 7, 15, 16 or 19. I l l 112 Figure 3.6 Percent s u r v i v a l of larvae from cont ro l l i n e 3 in fected with 14000 OBs of TnSNPV3 from (A) generations 0, 1, 2, 4, 5, 6, 10 and 15 (Assay 1) and; (B) generations 0, 5, 9, 11, 13 and 14 (Assay 2) . (C) Assays 1 and 2 combined. 113 (A) Assay 1 60 ^ 60 40 20 0 0 5 10 15 (B) Assay 2 0 5 10 15 (C) Assays combined 0 5 10 15 Generation 114 Figure 3.7 A-F R e s t r i c t i o n endonuclease p r o f i l e s of the DNA of the 5 TnSNPV i s o l a t e s : (1) o r i g i n a l TnSNPV for l i n e s 1 and 2, (2) TnSNPVl from generation 19, (3) TnSNPV2 from generat ion 19, (4) o r i g i n a l TnSNPV3, and (5) TnSNPV3 from generation 15 digested with e i ther (A) BamHI, (B) EcoRI, (C) H i n d l l l , (D) S s t I , (E) S a i l and (F) PstI and electrophoresed on a 0.7% agarose gel.9vphage DNA H i n d l l l r e s t r i c t i o n fragments were included as molecular weight markers. The s i z e of bands i s ind icated in k i lobase pa i rs and d i f fe rences i n p r o f i l e s are ind ica ted by white arrows. 115 A) BamHI _ 1 1 2 3 4 S | 23.1 9.4 6.5 2.3 • 116 o o 111 C%J f f f P t f f H Y ro CM A A A V ID AA ro o 117 x o fffPUfHV Oft COO A A A AA LT< CO CO o <0 csicsi 118 D) Pst I J 1 2 3 4 5 23.1 9.4 6.5 — 2.3 ! • 2.0 fr^ 119 23.1 9.4 6.5 4.3 2.3 2.0 E) SAL 1 T5 c: 1 2 3 120 23.1 9.4 6.5 4 . 3 F) Sst I T5 73 c . 9 5 1 2 3 4 5 2.3 ^ 2.0 | ^ 121 CHAPTER FOUR Is Resistance to the Singly-Embedded Nuclear Polyhedrosis Virus Of Trichoplusia n i Costly? Natural s e l e c t i o n cannot maximize the f i t n e s s of i n d i v i d u a l s with respect to a l l t r a i t s . When there i s a negative genet ic c o r r e l a t i o n between two characters , s e l e c t i o n w i l l favour an intermediate l e v e l for both t r a i t s (Futuyma 1986). Such compromises or t r a d e - o f f s could play an important r o l e i n the evolut ion of l i f e - h i s t o r y t r a i t s (Stearns 1992), and in the maintenance of genet ic v a r i a t i o n i n populat ions (e .g . Lande 1980, Rose 1982). Host-pathogen assoc ia t ions are be l ieved to be good systems to detect such t r a d e - o f f s . That i s because s e l e c t i o n w i l l favour hosts that can evolve res is tance to the pathogen. Resistance to pathogens i s however f requent ly assumed to be c o s t l y as i t may involve the d ive rs ion of energy from growth and/or reproduction to combatting the pathogen ( e . g . Simms and Rausher 1987, Simms and F r i t z 1990). For example, s e l e c t i o n could favour moderate l e v e l s of res is tance at the expense of s l i g h t l y reduced fecundi ty . Coevolutionary models 122 suggest that hosts w i l l evolve to a l e v e l of res is tance that i s determined by the cost of the res is tance (e .g . Mode 1958, G i l l e s p i e 1975, Simms and Rausher 1987). If res is tance i s not c o s t l y , then hosts w i l l evolve maximum r e s i s t a n c e . However, i f res is tance does e n t a i l any c o s t s , then i t w i l l be constrained to some intermediate va lue. In t h i s case, hosts w i l l evolve so as to maximize the d i f f e rence between the benef i ts and the costs associated with the res is tance (Simms and Rausher 1987). The conventional protocol used to assess the cost of res is tance cons is ts of comparing the f i t n e s s of r e s i s t a n t and suscept ib le i n d i v i d u a l s i n the absence of pathogens ( e . g . Simms and Rausher 1987, Groeters et a l . 1994, Simms and T r i p l e t t 1994, Biere and Antonovics 1996 see a lso Berenbaum et a l . 1986). If res is tance i s c o s t l y , r e s i s t a n t ind iv idua l s should have lower f i t n e s s than suscept ib le i n d i v i d u a l s i n pathogen-free environments. In Chapter 3, I observed that cabbage loopers (T r ichop lus ia n i ) could evolve res is tance to the s i n g l y -embedded nuclear polyhedrosis v i r u s of T. n i (TnSNPV). In t h i s chapter, I examine i f res is tance to TnSNPV i s c o s t l y by rear ing contro l and se lected l i n e s of T. n i i n the absence of v i r u s . I p red ic t that se lected l i n e s w i l l have prolonged 123 developmental t ime, reduced pupal weight and/or lower reproductive success than contro l l i n e s i f res is tance i s c o s t l y . Determining whether res is tance to TnSNPV i s c o s t l y has important p r a c t i c a l imp l i ca t ions . If i t does incur c o s t s , i t might be unstable and dec l ine once the v i r u s has been removed (e .g . Fuxa and Richter 1989 see a lso Osman et a l . 1991, Cochran 1993, Tabashnik et a l . 1991, 1994a). Thus, i t may be poss ib le to prevent or retard the evolut ion of res is tance to TnSNPV i n the f i e l d by using the v i r u s i n ro ta t ion with i n s e c t i c i d e s that have d i f f e r e n t modes of ac t ion (Georghiou 1983, Roush and Daly 1990). 124 MATERIALS AND METHODS At generation 20, I i n d i v i d u a l l y placed 40 neonates from each contro l and se lected l i n e s 1 and 2 in 25 ml p l a s t i c cups containing high wheat germ d i e t (Table 1.1) . The larvae were reared at 26±1°C on 16:8 (L:D) photoperiod and checked d a i l y . I recorded pupal weight (on the day of pupat ion) , sex and date of adult emergence. At generations 13-17, I i n d i v i d u a l l y reared 20-25 larvae from contro l and se lected l i n e 3 as descr ibed above. For generations 14-16, I recorded pupal weight, sex and the durat ion of the developmental time from egg to adult emergence. For generations 13-17, 8-10 p a i r s of adul ts from contro l and se lected l i n e 3 were mated in 500 ml paper cups (see Chapters 1 and 2) . The f i r s t paper l i n i n g was c o l l e c t e d four days a f te r a pa i r was formed and every second day u n t i l the female died ( t y p i c a l l y 4 l i n i n g s of paper) . For generation 16, I only c o l l e c t e d 3 l i n i n g s of paper per p a i r . At generation 16, I recorded the hatching success of eggs l a i d by females of contro l and se lected l i n e s 3 on the f i r s t l i n i n g of paper. At generation 17, I recorded the hatching success of a l l eggs l a i d by females. The eggs were not s u r f a c e - s t e r i l i z e d p r i o r to est imating hatching success as bleaching may adversely a f f e c t the v i a b i l i t y of eggs. 125 RESULTS Lines 1 and 2 The mean pupal weight of females and males from se lected l i n e s 1 and 2 was lower than that of cont ro l l i n e s 1 and 2 (see Figure 4 .1 ) . However, nei ther r e l a t i o n s h i p was c lose to s i g n i f i c a n c e (females: t = 1.070, df = 2, P = 0.199; males; t = 0.922, df = 2 , P = 0.454). The mean developmental time of females from se lected l i n e s 1 and 2 was shorter than those from contro l l i n e s 1 and 2 and the d i f fe rence was near ly s i g n i f i c a n t (see Figure 4.2; t = 2.668, df = 2, P = 0.058). The mean developmental time of males from cont ro l l i n e s 1 and 2 d id not d i f f e r from that of males of se lected l i n e s 1 and 2 (see Figure 4.2; t = 0.415, df = 2, P = 0.359). Lines 3 There was no d i f fe rence in the mean pupal weight of females from cont ro l and se lec ted l i n e s 3 at generation 14 (t = -0 .203, df = 13, P = 0.419) or 16 (Figure 4.3; t = 1.496, df = 27, P = 0.073). A l s o , there was no d i f fe rence in the mean pupal weight of cont ro l and se lected males at generation 14 (Figure 4.3; t = -0 .720, df = 23, P = 0.251) or generation 16 (t = -0 .577, df = 18, 126 P = 0.286). The developmental time of cont ro l and se lec ted males and females d id not d i f f e r at e i ther generation 14 (Figure 4.4; females: t = 1.257, df = 13, P = 0.115; males: t = -1 .043, df = 23, P = 0.154) or 16 (Figure 4.4; females: t = -0 .169, df = 27, P = 0.434; males: no var iance for se lec ted males) At generation 13, adult p a i r s from se lected l i n e 3 produced fewer eggs than those from cont ro l l i n e 3 (Figure 4.5; t = -2.294, df = 11, P = 0.042). However, there was no s i g n i f i c a n t d i f fe rence among contro l and se lected adul ts at generations 14 to 17 (Figure 4 .5; generat ion 14: t = 0.101, df = 12, P = 0.461; generation 15: t = 0.923, df = 15, P = 0.180: generation 16: t = 0.069, df = 16, P = 0.473; generation 17: t = 0.237, df = 14, P = 0.411). At generation 16, females from contro l l i n e 3 hatched twice as many eggs as se lected moths (Figure 4.6; t - t e s t on a r c s i n square root % hatching success; t = 2.771, df = 16, P = 0.018) but there was no d i f fe rence at generation 17 (Figure 4.6; t = 0.251, df = 14, P = 0.400). 127 DISCUSSION Costs of res is tance can occur because of p le io t ropy or l inkage among genes. With p le io t ropy , gene(s) that confer res is tance a lso adversely a f f e c t some other components of f i t n e s s (e .g . reproductive success) . With l inkage, gene(s) c o n t r o l l i n g res is tance are inher i ted with a l l e l e ( s ) that have negative e f fec ts on f i t n e s s more often that expected by chance (e .g . Falconer 1989). Resistance to TnSNPV d id not appear to incur many f i t n e s s c o s t s . F i r s t , the pupal weight and developmental time of contro l and se lected in d iv id u a ls d id not d i f f e r . This f i n d i n g i s consis tent with Chapter 1. The pupal weight and the developmental time of T. n i reared in the absence of v i r u s (Table 1.5) were independent of the res is tance (LD50) of t h e i r populat ions. Second, females from cont ro l l i n e 3 l a i d more eggs than those from se lected l i n e 3 in only 1 of 5 generations (generation 13). In that instance, the fecundi ty of females from contro l l i n e 3 was unusual ly h igh , over 1400 eggs. The fecundity of females from se lec ted l i n e 3 at generation 13 was about 1000 eggs, roughly the same number as females from cont ro l l i n e 3 at generations 14, 15 and 17. T h i r d , females from contro l l i n e 3 hatched more eggs than females from se lected l i n e 3 at generation 16 but there was 128 no d i f fe rence between females of cont ro l and se lec ted l i n e 3 at generation 17. Conversely, res is tance to baculoviruses has been shown to involve reproductive costs in Spodoptera f rugiperda (Fuxa and Richter 1989) and P lod ia in te rp u n c te l l a (Boots and Begon 1995). In both spec ies , r e s i s t a n t females l a i d and hatched fewer eggs than suscept ib le ones. In Chapter 1, I also observed that the mean egg production per female was lower in populat ions with high LD50s (Table 1.5) . I d i d not observe any re la t ionsh ip between a r c s i n square root % egg hatch and LD50 in Chapter 1. But i t i s i n t e r e s t i n g that in Chapter 1 the LD50 and % egg hatch (not transformed) were negat ive ly re la ted and f a i r l y c lose to s i g n i f i c a n c e (Pearson r = -0.438, N = 10, P = 0.100, 1 - t a i l p r o b a b i l i t y ) . Three explanations can be given to account fo r the d iscrepancies in the f ind ings of Chapters 1 and 4, and to expla in why cont ro l and se lec ted l i n e s may have been equal ly f i t . F i r s t , the negative r e l a t i o n s h i p between egg production and LD50 reported i n Chapter 1 may have been spur ious . The r e s u l t s of Chapter 4 may be more r e l i a b l e as they are based on f i v e separate experiments. Second, res is tance may i n i t i a l l y have been c o s t l y , but s e l e c t i o n for modif ier genes at other l o c i may have improved the harmful p l e i o t r o p i c 129 e f f e c t s of r e s i s t a n c e . For example, natura l populat ions of L u c i l l a cuprina that evolved res is tance to the chemical d iaz inon l a te r evolved compensatory changes to lessen the cost of res is tance (McKenzie et a l . 1982). S imi lar f ind ings have been observed i n b a c t e r i a (Pruss et a l . 1982, Oram and F isher 1992, Cohan et a l . 1994 see a lso Templeton 1980, Bouma and Lenski 1988). On the other hand, the o r i g i n a l res is tance with negative p l e i o t r o p i c e f f e c t s may have been replaced with an a l t e r n a t i v e mutation confer r ing the same res is tance but at a lower cost (Haldane 1932). T h i r d , i f the cost of res is tance observed in Chapter 1 was the r e s u l t of an assoc ia t ion between genes c o n t r o l l i n g res is tance and adversely a f f e c t i n g f i t n e s s , I may have broken the l inkages when I mixed the cabbage looper populat ions studied i n Chapter 1 to e s t a b l i s h the contro l and s e l e c t i o n l i n e s of the coevolut ion experiment (Chapter 3) . The r e s u l t s of t h i s study suggest that the cost and the l e v e l of res is tance may be independent in T. n i . Although se lected l i n e 3 evolved greater res is tance (RR = 25) than se lec ted l i n e s 1 and 2 (mean RR = 4.4; Chapter 3 ) , i t d i d not incur higher c o s t s , at l e a s t . i n terms of developmental time and pupal weight, a poss ib le cor re la te of egg product ion. The observat ion that res is tance to TnSNPV may not incur 130 any f i t n e s s costs i s consis tent with one of the f ind ings of Chapter 3. When s e l e c t i o n was d iscont inued, the mean LD50 of se lec ted l i n e s 1 and 2 d id not dec l ine markedly (Figure 3.3B). However, t h i s experiment should be repeated to determine i f l i n e s r e s i s t a n t to TnSNPV would revert to s u s c e p t i b i l i t y i f the v i r u s was removed for a longer per iod of t ime. The cost of res is tance i s be l ieved to p lay an important ro le in the maintenance of res is tance polymorphism ( e . g . Jarosz and Burdon 1991, Parker 1992). If res is tance i s not c o s t l y then a l l i nd iv idua ls w i l l become r e s i s t a n t and a l l of the var iance w i l l be exhausted. However, Antonovics and T h r a l l (1994) have shown that res is tance polymorphism can be maintained by very small c o s t s . The res is tance of cabbage loopers to TnSNPV may incur small costs that I was unable to detect . The costs of res is tance to TnSNPV may a lso be more pronounced in the f i e l d than in a con t ro l l ed laboratory s e t t i n g . The s i n g l e greatest t h e o r e t i c a l d i f f i c u l t y with s tudies on costs of res is tance i s that i t i s impossible to conclude that such costs do not occur . Resistance may incur subt le costs that the invest iga tor d id not examine. For example, res is tance to one pathogen may increase the s u s c e p t i b i l i t y of 131 the host to a second pathogen (Parker 1990, Simms and F r i t z 1990). This i s p a r t i c u l a r l y important in the present case as Fuxa and Richter (1990) showed that S. f rugiperda se lec ted for res is tance to NPV were more suscept ib le to methyl parathion than c o n t r o l s . In t h i s chapter, I observed that even cabbage loopers that exhib i ted 25 f o l d res is tance to TnSNPV d id not incur any costs i n terms of pupal weight, developmental t ime, egg product ion and probably egg v i a b i l i t y . F i n a l l y , from a p r a c t i c a l and t h e o r e t i c a l perspect ive i t i s important to determine i f the res is tance of cabbage loopers to TnSNPV a f f e c t s the s u s c e p t i b i l i t y of T. n i to other pathogens. If t h i s was the case, i t may be poss ib le to re tard or prevent the evolut ion of res is tance in the f i e l d by using TnSNPV in ro ta t ion with i n s e c t i c i d e s that have d i f f e r e n t modes of ac t ion (Georghiou 1983 and Roush and Daly 1990). 132 Figure 4.1 Mean (+1 SE) pupal weight of (A) females and (B) males from cont ro l and se lec ted l i n e s 1 and 2. 133 CD (A) Females "CD ZD CL C crj (B) Males o O CM i C o O "CD O CO CO 134 Figure 4.2 Mean (+1 SE) developmental time of (A) females (B) males from cont ro l and se lec ted l i n e s 1 and 135 CO CD (A) Females CTJ CD E C L O CD > CD Q CTJ CD (B) Males T - CM o O c o O i - CM CD CO CD CO 136 Figure 4.3 Mean (+1 SE) pupal weight of (A) females and (B) males from cont ro l (A) and se lec ted (•) l i n e s 3 at generations 14 and 16. 137 E (A) Females 'CD crj Q _ CL C Ctf CD (B) Males 14 16 Generation 138 Figure 4.4 Mean (+1 SE) developmental time of (A) females and (B) males from cont ro l (•) and se lec ted (•) l i n e s 3 at generations 14 and 16. 139 (A) Females CTJ 0 £ CTJ CD E Q_ O CD > 0 a c CTJ 0 (B) Males 14 16 Generation 140 Figure 4.5 Mean (+1 SE) eggs l a i d females (•) of l i n e s 3 by cont ro l (•) and se lec ted for generations 13-17. 141 Line 3 1600 -1400 H 1200 1000 800 600 H 12 13 14 15 16 17 18 Generation 142 Figure 4.6 Mean (+1 SE) percent egg hatch of eggs l a i d by cont ro l (A) and se lected (•) females of l i n e 3 at generations 16 and 17. 143 Line 3 100 80 60 40 16 17 Generation 144 CHAPTER FIVE The Effect of Larval Age on the Sublethal Effects of the Singly-Embedded Nuclear Polyhedrosis Virus of Trichoplusia n i on Cabbage Loopers Nuclear polyhedrosis v i ruses (NPVs) can a f fec t the demography of t h e i r lepidopteran hosts i n two ways. F i r s t , although inf luenced by the dose and the age at which c a t e r p i l l a r s get i n f e c t e d , NPVs can be l e t h a l . Young larvae have frequent ly been shown to be more suscept ib le to NPVs (e .g . Ignoffo 1966, Boucias and Nordin 1977, Sheppard and S t a i r s 1977, Burjeron et a l . 1981, Payne et a l . 1981, Teakle et a l . 1986, Smits and Vlak 1988). Larvae succumbing to the disease are e a s i l y recognized and the gross pathology of NPVs i s wel l documented (e .g . Mazzone 1985, Evans and Entwist le 1987). C a t e r p i l l a r s contract the v i r u s by ingest ing food contaminated with NPV. As the disease progresses, larvae stop feeding and become l e t h a r g i c , d i s c o l o r and eventual ly d i e . The cadavers are l imp, l yse e a s i l y and are f requent ly found hanging from branches or leaves in an inverted V p o s i t i o n . NPV ep izoot ics are commonly associated with c o l l a p s i n g 145 populat ions of fo res t c a t e r p i l l a r s ( e .g . Steinhaus 1949, B i r d and Burk 1961, Doane 1976, E lk in ton and Liebhold 1990 see Myers 1988 for a review). The age s t ruc ture of a populat ion i s an important determinant of the development and magnitude of v i r a l ep i zoot ics (Webb and Shelton 1990, Sa i t et a l . 1994a see a lso Onstad et a l . 1991). Second, although l e s s understood, NPVs may a lso have sublethal e f f e c t s . Indiv iduals surv iv ing nucleopolyhedrosis may su f fe r sub t le , chronic e f f e c t s . The p o s s i b l e de le ter ious e f fec ts of NPVs at enzootic l e v e l s may be of great importance in understanding the populat ion dynamics of the host ( e . g . Falcon 1971, P e r e l l e and Harper 1986, Rothman and Myers 1996 see a lso Ignoffo and Adams 1966, Anderson 1991, Onstad and et a l . 1991, Sa i t et a l . 1994a). Rothman and Myers (1996) synthesized the f ind ings of studies on suble tha l i n f e c t i o n s and came to four conclus ions: (1) i n d i v i d u a l s surv iv ing NPV i n f e c t i o n s during the l a r v a l stage are f requent ly impaired r e l a t i v e to c o n t r o l s . (2) The d e b i l i t a t i n g e f fec ts include prolonged development, reduced pupal weight and fecundi ty , shorter adult l i f e s p a n and lower hatching success of eggs. (3) The i n c l u s i o n of d e b i l i t a t i n g e f f e c t s caused by suble tha l i n f e c t i o n s of NPVs could reduce the growth of populat ions by an add i t iona l 22% over that of NPV-induced mor ta l i ty a lone. (4) The magnitude of d e b i l i t a t i n g e f f e c t s i s greater when 146 larvae are in fected as l a te ins ta rs (4 - 5th) but appears to be independent of the dose of v i r u s that larvae ingest (but see Sa i t et a l . 1994a, Shapiro et a l . 1994). Two major drawbacks associated with the use of NPVs as b i o l o g i c a l contro l agents are (1) they are expensive to produce and (2) the proport ion of i n d i v i d u a l s k i l l e d i s f requent ly lower than with chemicals. By determining which stage of the pest i s most suscept ib le to the d isease , i t may be poss ib le to ascer ta in when the v i r u s should be appl ied to maximize crop protec t ion while minimizing the dose requi red . Prec ise t iming of app l ica t ions could make NPV i n s e c t i c i d e s more cost e f f e c t i v e . In a d d i t i o n , demonstrating that surv ivors of nucleopolyhedrosis may be impaired and that these d e b i l i t a t i n g e f fec ts could adversely a f fec t the demography of the target insect may a id in making NPVs more e f f i c i e n t and marketable as i n s e c t i c i d e s . In t h i s chapter, I examined the i n t e r a c t i o n of TnSNPV dose and the age of T r i c h o p l u s i a n i c a t e r p i l l a r s at the time of i n f e c t i o n on the l e t h a l and suble tha l e f f e c t s of TnSNPV. S p e c i f i c a l l y , I ask the fo l lowing quest ions: Are young larvae more suscept ib le to the disease? Are in d iv id ua ls surv iv ing TnSNPV i n f e c t i o n impaired r e l a t i v e to cont ro l insects? Are these d e b i l i t a t i n g e f f e c t s a funct ion of the age 147 of the larvae at time of i n f e c t i o n and/or of the dose of v i rus? 148 MATERIAL AND METHODS The fo l lowing experiment was conducted twice under i d e n t i c a l p ro toco ls . In both experiments, I used insects from contro l l i n e 1 (see Chapter 3) and the same v i r u s preparat ion as in Chapters 1 and 2. T r i c h o p l u s i a n i eggs were surface s t e r i l i z e d as descr ibed in Chapter 1. Neonates were i n d i v i d u a l l y placed in 25 ml p l a s t i c cups containing a high wheat germ d i e t (Table 1.1) and allowed to feed for 96 (3rd) , 144 (4th) or 192 (5th instar ) hr at 26 C at 16:8 (L:D) photoperiod. Larvae were then in fec ted as descr ibed i n Chapter 1 with 2565, 12825 or 25650 TnSNPV OBs (10 u l of 2.565 x 10 , 1.283 x 10 or 2.565 x 10 , respect ive ly ) and each dose was r e p l i c a t e d 4 t imes. From the lowest to highest concentrat ion, the number of larvae per dose r e p l i c a t e was: 96 hr: 20, 25 and 35; 144 hr: 15, 18 and 20 (only 10 for dose 2565 OBs in T r i a l 2); 196 hr: 8, 8 and 12. I var ied the number of i n d i v i d u a l s per TnSNPV concentrat ion to ensure that there would be a s u f f i c i e n t number of surv iv ing adul ts in each age x dose category. For each age category, cohorts of 20 larvae were fed plugs t reated with d i s t i l l e d water as c o n t r o l s . Because 96 hr o l d c a t e r p i l l a r s eat slowly I allowed a l l c a t e r p i l l a r s 36 hr to consume the plug to be consis tent across the i n f e c t i o n age 149 groups. Larvae that consumed the en t i re plug wi th in 36 hr (98% of t reated ind iv idua ls ) were returned to t h e i r o r i g i n a l cup while those f a i l i n g to do so were d iscarded. The time to consume the plug (Appendix I) or s ta rva t ion for 48 hr fo l lowing ingest ion of the plug does not a f f e c t the s u s c e p t i b i l i t y of larvae to TnSNPV (M. M i l k s , unpublished data) . Larvae were checked d a i l y and mor ta l i t y , pupal weight (on day of pupation) and time of adult emergence were recorded. Diagnosis of v i r a l mor ta l i ty was based on gross symptoms. O v e r a l l , 96% of TnSNPV inoculated larvae that died exhib i ted the t y p i c a l s igns of nucleopolyhedrosis (Evans and Entwist le 1987). Control mor ta l i ty never exceeded 5%. Seven randomly se lected female and male moths from each treatment were pai red and placed in 500 ml L i l y paper cups (see Chapters 1, 2 and 4; i . e . one p a i r / c u p , 7 cups x 3 l a r v a l age x 4 TnSNPV treatment [3 v i r u s doses + 1 contro l ] = 84 cups) . The f i r s t paper l i n i n g was c o l l e c t e d 4 days a f te r a pa i r was formed and every second day thereaf ter u n t i l the female d i e d . The number of eggs per l i n i n g was counted. In T r i a l 2, I a lso estimated the hatching success of eggs on the f i r s t l i n i n g of paper. The equation of the dosage-mortal i ty curves and LD50s with associated 95% confidence i n t e r v a l s of each l a r v a l 150 age x TnSNPV treatment fo r T r i a l s 1 and 2 (3 age groups per t r i a l x 2 t r i a l s = 6 curves) were computed using a prob i t model (PROC PROBIT, SAS Ins t i tu te 1990). The heterogeneity chi -square values were small (P > 0.05) for 5 of the 6 curves i n d i c a t i n g that the observed mor ta l i ty f i t t e d a prob i t model (Finney 1971). The slopes and in tercepts of the dosage-mortal i ty curves were compared wi th in and among t r i a l s as descr ibed in Chapter 1. The poss ib le suble tha l e f f e c t s of TnSNPV on developmental time, pupal weight and reproductive success (number of eggs l a i d per female and hatching success of eggs) were examined using analyses of var iance (PROC GLM, SAS 1990). The independent va r iab les were TnSNPV treatment (3 TnSNPV doses + 1 contro l = 4 l e v e l s , f i xed e f f e c t ) , l a r v a l age at i n f e c t i o n (3 l e v e l s , f i xed e f f e c t ) , sex (2 l e v e l s , f i xed e f fec t ) and t r i a l (2 l e v e l s , random e f f e c t ) . Data were pooled across TnSNPV r e p l i c a t e s . I i n i t i a l l y estimated a saturated model i . e . a model that incorporated a l l main e f f e c t s and p o s s i b l e i n t e r a c t i o n terms. I then sequent ia l l y removed the highest l e v e l i n t e r a c t i o n term that had the highest p r o b a b i l i t y value ( least s i g n i f i c a n t ) from the model and re-est imated the ANOVA. However, I d id not remove a low l e v e l term i f a higher 151 l e v e l term was s i g n i f i c a n t . For example, i f A x B x C was s i g n i f i c a n t then A x B, A x C and B x C would remain in the model even though they might not be s i g n i f i c a n t . I repeated t h i s process u n t i l I could not remove any more terms from the model (= reduced model). I adopted t h i s strategy i n order to obtain the most parsimonious model. 152 RESULTS Surv iva l of larvae inoculated with TnSNPV at d i f f e r e n t ages The slope and in tercept of dosage-mortal i ty curves both var ied with the age of larvae at i n f e c t i o n i n T r i a l s 1 and 2 (Table 5.1; a l l chi -squares >_ 9.2, df = 2, P < 0.01). The slope of the dosage-mortal i ty curve for larvae inoculated at 4-days-of -age was steeper i n T r i a l 1 than i n T r i a l 2 (Table 5.1; chi -square = 6.58, df = 2, 0.025 < P < 0.05). Otherwise, the slopes and in tercepts wi th in a given age group d i d not d i f f e r across t r i a l s . Percent s u r v i v a l to adulthood and LD50 increased with the age at which c a t e r p i l l a r s were in fected in a l l but one case (Table 5.1) . I t was not poss ib le to estimate 95% confidence l i m i t s fo r the LD50 of larvae in fected at 8-days-of-age i n T r i a l 1 because s u r v i v a l was greater than 69% for a l l doses. The s u r v i v a l to adulthood in each age by TnSNPV treatment (3 TnSNPV doses + 1 contro l ) was s i m i l a r i n both t r i a l s (Table 5.1) . Pupal weight of i n d i v i d u a l s surv iv ing to adulthood The saturated and reduced models for pupal weight d id 153 not d i f f e r q u a l i t a t i v e l y ; the four main e f f e c t s and the dose x age and age x t r i a l in te rac t ion terms were s i g n i f i c a n t (Table 5 .2) . To determine i f the pupal weight of surv ivors var ied among the 3 TnSNPV doses, I dropped the cont ro l groups and re-est imated the model. Here, ne i ther the main e f f e c t of dose nor the dose x age in te rac t ion term were s i g n i f i c a n t (Table 5 .2) . This suggests that mean pupal weight d id not vary among the 3 TnSNPV doses. Hence, in the f i n a l model, I pooled the 3 v i r u s concentrat ions and compared the pupal weight of ind iv idua ls surv iv ing i n f e c t i o n to cont ro ls T. n i . This model suggests that pupal weight was a f fected by three f a c t o r s : F i r s t , female pupae were l i g h t e r than males (Table 5.2, F igure 5.1) . Second, the weight of pupae var ied between t r i a l s fo r c a t e r p i l l a r s t reated at 6- and 8-days-of-age (Table 5.2 Figure 5.1) . However, the e f f e c t was not cons is ten t . Male and female pupae in the 6-day group were heavier i n T r i a l 1 than T r i a l 2, whereas the reverse was observed for the 8-day group ( t - t e s t on means adjusted for the e f f e c t of TnSNPV; 6-days: females, t = 4.38, df = 262, P < 0.001; males, t = 2.57, df = 211, P = 0.005; 8-day; females, t = 3.38, df = 262, P < 0.001; males, t = 2.19, df = 88, P = 0.015). T h i r d , the e f f ec t of TnSNPV i n f e c t i o n on the pupal 154 weight of surv ivors was a funct ion of the age at which the larvae were in fected (Table 5.2, Figure 5.1) . Time to adult emergence The saturated and reduced model for time to adult emergence were s i m i l a r ; the main e f f e c t s of TnSNPV dose, l a r v a l age at i n f e c t i o n , sex, and the age x t r i a l in te rac t ion term were s i g n i f i c a n t i n both the reduced and saturated models (Table 5.3) . T r i a l and dose x age were a lso s i g n i f i c a n t i n the reduced model (Table 5 .3) . To determine i f the time to adult emergence var ied among the 3 v i r u s concentrat ions, I dropped the cont ro l groups and re-est imated the model. Neither the main e f fec t nor in te rac t ion terms invo lv ing dose were s i g n i f i c a n t in t h i s model (Table 5 .3) . Th is suggest that time to adul t emergence d i d not vary among the three TnSNPV doses. Hence, as for pupal weight, I pooled the 3 TnSNPV doses. This model suggested that time to adult emergence was inf luenced by three f a c t o r s . F i r s t , female moths emerged before males (Table 5.3 and Figure 5.2) . Second, developmental time var ied across t r i a l s in some age groups (Table 5.3) . Males t reated at 4-and 6-days of the l a r v a l stage emerged sooner i n T r i a l 1 than T r i a l 2 ( t - t e s t on means adjusted for the e f fec t of TnSNPV treatment; 4-days: 155 t = -2 .03 , df = 208, P = 0.04; 6-days: t = - 2 .91 , df = 114, P = 0.004). Females fol lowed s i m i l a r trends but the r e l a t i o n s h i p was never s i g n i f i c a n t . T h i r d , the e f f ec t of TnSNPV i n f e c t i o n on developmental time was a funct ion of the age at which the larvae were in fected (Table 5.3, Figure 5.2) . Egg product ion The saturated and reduced model fo r the number of eggs l a i d per female were s i m i l a r ; the main e f f e c t s of l a r v a l age at i n f e c t i o n and t r i a l were s i g n i f i c a n t (Table 5.4) . I opted not to removed the dose x age in te rac t ion term from the reduced model because (1) i t was c lose to s i g n i f i c a n c e (P = 0.069, Table 5.4) and (2) because t h i s term was s i g n i f i c a n t i n the analyses of pupal weight and pupal weight i s cor re la ted with egg production in many species of i n s e c t s . To be consis tent with my strategy when modeling pupal weight and developmental time, I removed the cont ro l groups and re -estimated the reduced model. The p r o b a b i l i t y values of the main e f f e c t of dose and the in te rac t ion term of dose x age both increased considerably (Table 5 .4) . Hence, i n the f i n a l model, I pooled the 3 TnSNPV doses and compared the egg 156 production of i n d i v i d u a l s surv iv ing i n f e c t i o n to cont ro l T. n i . This f i n a l model suggested that the number of eggs l a i d per female was inf luenced by two f a c t o r s . F i r s t , females l a i d more eggs in T r i a l 1 ( t - t e s t on means adjusted for the e f f e c t of TnSNPV dose; 4-days, t = 2.64, P = 0.004; 6-days: t = 4.31, P < 0.001; 8 day, t = 3.34, P < 0.001). Second, the e f f e c t of TnSNPV i n f e c t i o n on egg product ion was a funct ion of the age at which larvae were t reated (Table 5.4, Figure 5 .3) . Hatching success of eggs No fac tor entered the saturated or reduced models descr ib ing the hatching success of eggs. When I pooled the 3 v i r u s concentrat ions, the d i f fe rence between in fected and cont ro l groups approached s i g n i f i c a n c e (P = 0.052) and the hatching success of eggs from in fected mothers was approximately 30% less than that of cont ro ls when T. n i are in fected at 4- and 6-days-of-age (Figure 5.4) . 157 DISCUSSION S u s c e p t i b i l i t y of larvae t reated at d i f f e r e n t ages O v e r a l l , s u r v i v a l and LD50 increased with the age at which c a t e r p i l l a r s were in fec ted (Table 5 .2) . Decl ine in s u s c e p t i b i l i t y to baculovi ruses with l a r v a l age at i n f e c t i o n has been reported i n H e l i o t h i s zea and H. v i rescens (Ignoffo 1966), Hyphantria cunea (Boucias and Nordin 1977), Cydia pomonella (Sheppard and S t a i r s 1977), Lymantria d ispar , Mamestra brass icae and Spodoptera l i t o r a l i s (Burgeron et a l . 1981, Evans 1981, 1983), H. punctiger (Teakle et a l . 1986), Sp. exigua (Smits and Vlak 1986), P lod ia in t e rpunc t e l l a (Sai t et a l . 1994b) and in many other species (see references in Ignoffo 1966, Boucias and Nordin 1977, Hochberg 1991). In some cases, dec l ine in s u s c e p t i b i l i t y has been explained by increase i n body weight. As larvae get o lder and b igger , there i s a greater mass of t i ssue to be in fec ted and a larger inoculum of v i r u s i s required to k i l l the host insec t (Ignoffo 1966). However, there are a lso many instances where the LD50/body weight s t i l l increases with age at i n f e c t i o n suggesting that increase in weight alone cannot e n t i r e l y account for the lower s u s c e p t i b i l i t y of o lder la rvae . Such supraproport ional decrease in s u s c e p t i b i l i t y (Sai t et a l . 1994b) appears to be more frequent in species with gregarious 158 feeding habi ts where the t ransmission rate of the disease may be high (Hochberg 1991). D e b i l i t a t i n g e f f e c t s of TnSNPV fo l lowing suble tha l i n f e c t i o n s Cabbage loopers that survived i n f e c t i o n with TnSNPV were impaired r e l a t i v e to cont ro ls i n severa l ways. Ind iv iduals that survived the disease had longer developmental t ime, reduced pupal weight, produced fewer eggs and hatched fewer eggs than contro ls (Figures 5.1 - 5.4) . In fect ion with NPVs or granulos is v i ruses have been shown to adversely a f f e c t developmental t ime, pupal weight, egg production and/or the hatching success of eggs of L. d i s p a r , Phtorimaea o p e r c u l e l l a , Sp. o r n i t h o g a l l i . Sp. l i t t o r a l i s , Malacosoma ca l i fo rn icum p l u v i a l e , Pseudola te l ia separata, S t i l p n o t i a s a l i c i s and P. in te rpunc te l l a (see Rothman and Myers 1996 for a review). Sa i t et a l . (1994a) out l ined two mechanisms to expla in how subletha l doses of v i r u s could adversely a f fec t the f i t n e s s of surv iv ing i n d i v i d u a l s . In the f i r s t , energy that i s used for growth or reproduction i s d iver ted to combat the disease (see Wiygul and Sikorowski 1978, 1991). The second poss ib le mechanism suggests that the v i r u s i s c a r r i e d in a form that does not k i l l the host (see Mazzone 1985; asymptomatic in fec t ions with NPVs) but can nevertheless 159 d isrupt metabolism (see Sikorowski and Thompson 1979) and oocyte development, damage t i ssues such as the fa t body or a l t e r hormone l e v e l s (Si lhacek and Oberlander 1975, Subrahmanyam and Ramakrishnan 1980, O ' R e i l l e y and M i l l e r 1989, Burand and Park 1992, O ' R e i l l e y 1995, ). Sp_. l i t o r a l i s (Saldanha and Hunter 1985) and M. brass icae (Hughes et a l . 1993) may carry baculoviruses without e x h i b i t i n g any symptoms of d isease . With t h i s type of mechanism, the magnitude of d e b i l i t a t i n g e f fec ts may be greater when i n d i v i d u a l s are in fected at a young age because they w i l l carry the disease for a longer per iod of t ime. The d e b i l i t a t i n g e f f e c t s r e s u l t i n g from suble tha l in fec t ions of TnSNPV d id not appear to be dose-dependent. When I dropped the cont ro l groups from the ANOVAs, the e f f e c t of dose (main and in te rac t ion with other fac tors ) was never s i g n i f i c a n t . In t h e i r review, Rothman and Myers (1996) concluded that there was l i t t l e evidence for the sublethal e f f e c t s of TnSNPV to be dose-dependent (but see Sa i t et a l . 1994a). The d e b i l i t a t i n g e f f e c t s r e s u l t i n g from subletha l i n f e c t i o n s of TnSNPV d i d , however, appear to be a funct ion of the age of the larvae at the time of i n f e c t i o n . Contrary to previous studies (see Rothman and Myers 1996 for a review), 160 the suble tha l e f f e c t s of TnSNPV were greatest when T. n i were in fected as young rather than la te i n s t a r s . The sublethal e f f e c t s of TnSNPV were strongest and most consis tent when larvae were inoculated at 4-days-of-age (Figure 5 . 1 - 5 . 4 ) . Cabbage loopers may not f i t the paradigm of Rothman and Myers ( d e b i l i t a t i n g e f f e c t s are most common when la te ins ta rs are infected) because T. n i have a short l a r v a l stage (11-13 days at 2 6 ° C ) . Thus, the v i r u s may not have enough time to have an impact when, the larvae are inoculated at 8 -days-of -age. D e b i l i t a t i n g e f f e c t s fo l lowing i n f e c t i o n of f i f t h ins ta rs has been observed in M. ca l i fo rn icum p l u v i a l e and PI . i n t e r p u n c t e l l a . In those spec ies , the durat ion of f i f t h ins ta r i s twice as long as in T. n i . Pupal weight and egg production per female are cor re la ted i n many species of i n s e c t s . In s p i t e of t h i s , the magnitude of d e b i l i t a t i n g e f f e c t s on fecundi ty was greater than on pupal weight. Females surv iv ing TnSNPV when t reated at 4-days-of-age l a i d 40% fewer eggs (average across both t r i a l s ) but , on average, only weighed 6 - 10% less than cont ro l females. This suggests that d i f fe rences in egg product ion per female cannot be e n t i r e l y explained by reduct ion in the pupal weight of females. Rothman and Myers (1994) a lso observed for M. ca l i fo rn icum p l u v i a l e that the 161 fecundity of in fec ted females was lower than cont ro ls even when v a r i a t i o n in pupal weight was s t a t i s t i c a l l y removed. They proposed that the add i t iona l reduct ion in egg product ion may have occurred because the v i r u s a f fec ted reproductive t i s s u e s . Sa i t et a l . (1994a) c r i t i c i z e d the use of the d i e t contamination technique, as the one used in t h i s study, when studying sublethal e f f e c t s of bacu lov i ruses . Their c r i t i c i s m res ts on the assumption that i n d i v i d u a l s that feed and develop more slowly may ingest the v i r u s over an extended per iod of time and have a higher p r o b a b i l i t y of s u r v i v a l . Thus, the inocu la t ion technique per se could s e l e c t fo r smal ler , slower developing i n d i v i d u a l s . I examined t h i s assumption in Appendix I and observed that (1) the majori ty of c a t e r p i l l a r s f i n i s h e d eat ing the plug f a i r l y synchronously and (2) the ingest ion time of the plug d id not a f f e c t s u r v i v a l . Ignoffo (1966) and V a i l and H a l l (1969) a lso examined the sublethal e f f e c t s of TnSNPV on T. n i but d id not observe any e f f e c t s when f i r s t or second ins ta rs were t rea ted . My f ind ings may d i f f e r for severa l reasons. F i r s t , although subletha l e f f e c t s have been reported f requent ly , they have not been observed in a l l s tudies (Rothman and Myers 1996). 162 In t h i s respect , the o v e r a l l f ind ings for cabbage loopers are consistent with the l i t e r a t u r e on d e b i l i t a t i n g e f f e c t s . The reason why d e b i l i t a t i n g e f f e c t s occur s p o r a d i c a l l y i s unknown. The magnitude of sublethal e f f e c t s may be a funct ion of the physiology of the insect at time of i n f e c t i o n . Engelhard and Volkman (1995) showed that jus t a few hours d i f fe rence in the age of c a t e r p i l l a r s can have dramatic e f f ec ts on the s u s c e p t i b i l i t y to T. n i to Autographa c a l i f o r n i c a NPV. The expression of subletha l e f f ec ts may a lso be under such t i g h t p h y s i o l o g i c a l c o n s t r a i n t s . In both experiments, larvae were in fected at the same age ( ± 1 h r ) . A second poss ib le explanat ion for v a r i a t i o n in r e s u l t s among studies i s that d e b i l i t a t i n g e f f e c t s may occur mostly with t h i r d ins ta rs in T. n i . Young ins ta rs are h igh ly suscept ib le to the disease and only those that escape i n f e c t i o n because of mechanical (pipet ing) e r rors may surv ive . Thus, in t h i s case, no d i f fe rence would be expected between contro l and pu ta t ive ly t reated i n d i v i d u a l s . Older larvae may only suf fe r minor d e b i l i t a t i n g e f fec ts because the v i r u s may not have enough time to es tab l ished i t s e l f (see above). The expression of sublethal e f f e c t s may a lso be under genet ic cont ro l and may vary from c a t e r p i l l a r to the other . The cabbage loopers used in t h i s study came from 5 d i f f e r e n t populat ions (see Mater ia ls and Methods) and may have been 163 more g e n e t i c a l l y d iverse than those used by Ignoffo (1966) and V a i l and Ha l l (1969). Impl icat ions of f ind ings for b i o l o g i c a l cont ro l The r e s u l t s of t h i s study suggest that a p p l i c a t i o n of TnSNPV when T. n i are i n the ea r ly stages of development may r e s u l t i n more e f f i c i e n t cont ro l of cabbage loopers fo r two reasons: (1) young larvae are more suscept ib le to the d isease; (2) surv ivors may have impaired development and reproduct ive success. These subletha l e f f e c t s could have add i t iona l adverse e f f e c t s on the demography of cabbage loopers . 164 Table 5.1 Percent survival, LD50 and slope and intercept of dosage-mortality curves in relation to the age at which T. ni larvae were inoculated. Trial Age of larvae at infection (days) 6 8 Mean % Survival (SE) 2565 OBs 1 2 12825 OBs 1 2 25650 OBs 1 2 80(3 74(1 36(1 30(1 22(1 13(1 86(3) 85(2) 62(5) 60(3) 41(1) 38(4) 91(1) 95(2) 69(1) 77(3) 78(3) 38(3) LD50 [95% C. I.] 1 9403 18625 [7294,11613] [13289,30354] 6016 [4424,7698] 20659 606142 [•••] 19839 [14235,38241] [14001,32194] Slope(SE) 1 2 3.06(0.39) 2.80(0.35) 2.07(0.44) 1.11(0.46) 0.30(0.54) 1.64(0.37) Intercept(SE) 1 -12.15(1.60) -8.83(1.81) -2.36(2.22) 2 -10.57(1.39) -4.47(2.56) -7.06(1.55) 165 Table 5.2 Four-way ANOVA of pupal weight with TnSNPV dose (Dose), larval age at infection (Age), sex (Sex) and Trial (Trial) as predictor variables. Factor Saturated Reduced Reduced Reduced Model Model Model Model Without Doses Control Pooled Dose 0.005 0.009 0.147 <0.001 Age <0.001 <0.001 0.001 <0.001 Trial 0.011 0.005 0.041 0.006 S e x <0.001 <0.001 <0.001 <0.001 Dose*Age 0.007 0.002 0.134 <0.001 Dose*Trial 0.300 Dose*Sex 0.320 Age*Trial <0.001 <0.001 <0.001 <0.001 Age*Sex 0.961 Trial*Sex 0.920 Dose*Age*Trial 0.406 Dose*Age*Sex 0.916 Dose*Trial*Sex 0.863 Age*Trial*Sex 0.361 Dose*Age*Trial*Sex 0.461 166 Table 5.3 A n a l y s i s of v a r i a n c e re la t i ng T n S N P V d o s e ( D o s e ) , l a r v a l a g e at in fect ion ( A g e ) , S e x ( S e x ) a n d T r i a l (Trial) to t i m e to adu l t e m e r g e n c e . F a c t o r S a t u r a t e d R e d u c e d R e d u c e d R e d u c e d M o d e l M o d e l M o d e l M o d e l W i t h o u t D o s e s C o n t r o l P o o l e d D o s e 0 . 0 1 6 0 . 0 0 7 0 . 8 0 7 <0 .001 A g e <0 .001 <0 .001 <0 .001 <0.001 T r i a l 0 . 2 2 3 0 . 1 6 5 0 . 8 3 8 0 . 0 0 6 S e x <0 .001 <0 .001 <0 .001 <0 .001 D o s e * A g e 0 . 1 0 7 0 . 0 5 3 0 . 2 9 2 0 . 0 2 8 D o s e * T r i a l 0 . 0 7 1 0 . 0 3 8 0 . 1 0 0 D o s e * S e x 0 . 1 6 7 A g e * T r i a l 0 . 0 1 6 0 . 0 0 1 7 0 . 0 6 4 <0 .001 A g e * S e x 0 . 5 6 6 T r i a l * S e x 0 . 7 0 0 D o s e * A g e * T r i a l 0 . 4 0 6 D o s e * A g e * S e x 0 . 9 1 6 . D o s e * T r i a l * S e x 0 . 8 6 3 A g e * T r i a l * S e x 0 . 3 6 1 D o s e * A g e * T r i a l * S e x 0 . 4 6 0 167 Table 5.4. Ana lys is of var iance relating T n S N P V dose (Dose), larval age at infection (Age) and trial (Trial) to the number of eggs laid per females. Factor Saturated R e d u c e d R e d u c e d R e d u c e d M o d e l M o d e l M o d e l M o d e l Without D o s e s Contro l P o o l e d D o s e 0 .088 0 .093 0 .978 0 .012 A g e 0 .010 0.011 0 .002 0 .430 Trial 0.001 <0.001 <0.001 <0.001 D o s e * A g e 0.062 0 .069 0.293 0 .044 Dose*Tr ia l 0 .748 A g e T r i a l 0 .247 Dose*Age*Tr ia l 0 .254 168 Figure 5.1 Adjusted mean (+1 SE) pupal weight (mg) of (A) females and (B) males surv iv ing to adulthood in r e l a t i o n to TnSNPV treatment and the age in days at which larvae were inocu la ted . Control -4 = ind iv idua ls mock in fected at 4-days-o l d of the l a r v a l stage. Infected-4 i s a pooled category denoting a l l i nd iv idua ls that were t reated with v i r u s i . e . 2565, 12825 or 25650 OBs of TnSNPV. Adjusted means ind ica tes that the averages have been adjusted for other covar ia tes (sex and t r i a l ) . 169 CD E CD CD 260 240 220 200 180 (A) Females ~\ r T r T r CO Q . Q_ c CO CD CD -*—> CO < 260 240 220 H 200 H 180 4 (B) Males "5 c o O T 3 CD +-> O CD CD CD • i O ~ ° id CD c o O c o CD 00 op C CD c o O CD O c 170 Figure 5.2 Adjusted mean developmental time (days) of (A) females and (B) males surv iv ing to adulthood i n r e l a t i o n to TnSNPV treatment and the age in days at which larvae were inocu la ted . Contro l -4 = ind iv idua ls mock in fected at 4 -days-o ld of the l a r v a l stage. Infected-4 i s a pooled category denoting a l l i nd iv idua ls that were t reated with v i r u s i . e . 2565, 12825 or 25650 OBs of TnSNPV. Adjusted means ind icates that the averages have been adjusted for other covar ia tes (sex and t r i a l ) . 171 21.0 -{ (A) Females CO 0 E CO c 0 E Q _ _o CD > 0 Q c CO 0 "D 0 •4—• V) U < (B) Males • c o O CD o 0 CD CD op trol-ted- o Conl nfecl Conl "O CD o CD 172 Eigure. 5.3 Mean. egg.. -production., per. female, in. r e l a t i o n to TnSNPV treatment, and the age. i n days at which larvae were inocu la ted , Contro l -4 = i n d i v i d u a l s mock in fec ted at 4 -days-o ld of the l a r v a l s tage . Infected-4 i s a pooled category denoting a l l i n d i v i d u a l s that were t reated with,, v i r u s i.».e» 2565, 12825 or 25650 OBs of TnSNPV. Adjusted means ind ica tes that the averages have been adjusted for other covar iates ( t r i a l ) . 173 o '•*-> o "D O C O C O LU CO 0 1400 1200 1000 o c o O "D 0 O 0 CD Cp -t—> -*—> c o O 0 O c 00 00 bf 0 •*—> •*—> c o O 0 O c 174 Figure 5.4 Mean % egg hatch in r e l a t i o n to TnSNPV treatment and the age i n days at which larvae were inocula ted . Control -4 = i n d i v i d u a l s mock in fected at 4 -days-o ld of the l a r v a l stage. Infected-4 i s a pooled category denoting a l l i n d i v i d u a l s that were t reated with v i r u s i . e . 2565, 12825 or 25650 OBs of TnSNPV. 175 100 -I 80 X co iff 60 CD O CD C L c crj CD 40 20 1 0 H o c o O "O CD •*—> O CD C D C D ~5 ~° CD •4—» -4—» c o O CD O c op op 75 ~& -t—> d o o o O c 176 CONCLUSIONS The success of nuclear polyhedrosis v i r u s e s (NPVs) as long-term pest cont ro l agents w i l l depend on the coevolut ion of the host and the v i r u s . In t h i s study, I used cabbage loopers (T r ichop lus ia n i ) as a lepidopteran model to conduct a v a r i e t y of experiments to examine d i f f e r e n t aspects of insect-NPV coevolu t ion . Summary of f ind ings In chapters 1 and 2, I observed that the s u s c e p t i b i l i t y of cabbage looper larvae to the singly-embedded nuclear polyhedrosis of T . n i (TnSNPV) var ied wi th in and among l i n e s of T. njL and that part of t h i s phenotypic v a r i a t i o n was g e n e t i c a l l y determined. These f ind ings suggest that cabbage loopers have the po ten t i a l to evolve res is tance to TnSNPV. In Chapter 3, I conducted two coevolut ion experiments and confirmed these f i n d i n g s . The s u r v i v a l of se lec ted larvae fed coevolved v i r u s increased from 10-20% to about 50% at the conclusion of s e l e c t i o n . Assays fo r the res is tance of T. n i with the o r i g i n a l v i r u s showed that the mean LD50 of se lec ted l i n e s 1 and 2 was 4.4 times greater and that of se lec ted l i n e 3 was 25 times greater than t h e i r cont ro l counterparts . Selected l i n e 3 may have evolved higher res is tance than the 177 other two se lected l i n e s because i t may have been more g e n e t i c a l l y d iverse at the onset of s e l e c t i o n . However, mainly because of t echn ica l problems associa ted with the counting of occ lus ion bodies , i t i s d i f f i c u l t to make d e f i n i t e conclusions about the evolut ion of the v i r u s . TnSNPV-1 and Z2L d id not appear to evolve higher v i r u l e n c e . However, some evidence suggests that TnSNPV-3 may have become more v i r u l e n t : (1) i n the assays for v i r u l e n c e , the s u r v i v a l of c a t e r p i l l a r s from cont ro l l i n e 3 dec l ined with the generation from which the v i r u s was i s o l a t e d ; (2) although T. n i from se lec ted l i n e 3 evolved 25 f o l d r e s i s t a n c e , 14000 OBs of coevolved TnSNPV3 was capable of k i l l i n g approximately 50% of se lected larvae at the conclusion of the experiment. Thus, i t seems that TnSNPV would have had to evolve higher v i ru lence to s t i l l be able to k i l l ha l f of the larvae exh ib i t ing such high r e s i s t a n c e . Se lec t ion may have favoured increased v i ru lence for TnSNPV-3 because c a t e r p i l l a r s of se lected l i n e 3 evolved considerably higher res is tance than those of se lected l i n e s 1 and 2 (see previous paragraph). Hence, the evolut ion of TnSNPV v i ru lence may be t i g h t l y associated and determined by the evolut ion of res is tance in T. n i -O v e r a l l , my r e s u l t s suggest that res is tance to TnSNPV 178 may incur few c o s t s . The f ind ings of chapters 1 and 4 suggest that res is tance to TnSNPV does not a f f e c t the developmental time and pupal weight of cabbage loopers . The e f f e c t of res is tance to TnSNPV on reproductive success i s more c o n t r o v e r s i a l . In Chapter 1, res is tance (LD50) was negat ive ly corre la ted with the number of eggs l a i d per female but i t was not cor re la ted with the hatching success of eggs. In Chapter 4, however, T . n i se lec ted for res is tance produced as many eggs as contro ls in 4 of 5 experiments but hatched fewer eggs in 1 of 2 experiments. If res is tance to TnSNPV does not incur any f i t n e s s c o s t s , then cabbage loopers should evolve maximum res is tance when cont inuously exposed to the d isease . The success of TnSNPV as a long-term pest cont ro l agent would thus depend on the v i r u s evolv ing higher v i ru lence to compensate for the res is tance of the host . When the v i r u s i s used as a short- term pest cont ro l agent ( i . e . l i k e chemicals) , i t might s t i l l be poss ib le to manage the evo lu t ion the res is tance i f res is tance to TnSNPV increases the s u s c e p t i b i l i t y of cabbage loopers to other pathogens (see Fuxa and Richter 1990). 179 Impl icat ions of r e s u l t s fo r f i e l d populat ions of insec ts Although t h i s study was conducted in the laboratory , i t should be poss ib le to extrapolate these f i n d i n g s , at l eas t q u a l i t a t i v e l y , to the f i e l d . The s u s c e p t i b i l i t y of cabbage loopers to TnSNPV i s l i k e l y to vary among and wi th in wi ld populat ions of T. ni^. The h e r i t a b i l i t y of s u s c e p t i b i l i t y i n the f i e l d could be even higher than that ca lcu la ted i n Chapter 2. Cabbage loopers are strong migrants and capable of long-distance f l i g h t s (Mi tche l l and Chalfant 1984). Thus, a newly sowed cabbage patch may be colonized by cabbage loopers from a v a r i e t y of sources. Thus, there could be more v a r i a t i o n in s u s c e p t i b i l i t y among i n d i v i d u a l s in nature than there was in my experiments i n Chapter 2. Wild populat ions of cabbage loopers should a lso be able to evolve res is tance to TnSNPV when cont inuously exposed to the v i r u s . However, the magnitude and the rate of evolut ion of res is tance in the f i e l d may d i f f e r from that in the laboratory for several reasons. F i r s t , varying temperature r e l a t i v e humidity and other c l i m a t i c f a c t o r s , the presence of other pathogens and the i n a c t i v a t i o n of NPV by u l t r a -v i o l e t l i g h t could a f f e c t the evo lu t ion of r e s i s t a n c e . Second, cabbage loopers are more suscept ib le to the disease when the v i r u s i s administered to larvae on leaf d i s c s than 180 on plugs of a r t i f i c i a l d i e t (M. M i l k s , unpublished data ) . T h i r d , the s u s c e p t i b i l i t y of cabbage loopers a lso var ies with host p lant (M. M i l k s , unpublished data) , and thus res is tance may evolve fas ter on some crops than others . Fourth, fo l lowing the co l lapse of a populat ion because of a v i r a l e p i z o o t i c , the area may act as a sink and the immigration of suscept ib le ind iv idua ls could slow the evolut ion of res is tance . F i f t h , res is tance to TnSNPV may evolve fas te r in some par ts of the cabbage looper range. For example, T. n i has 12 generations per year in the F l o r i d a panhandle but only 1-3 per year i n B r i t i s h Columbia (Mi tche l l and Chalfant 1984). Moreover, populat ions in the northernmost part of the cabbage looper range and bel ieved to be r e - e s t a b l i s h e d every year by migrants from the south (Mi tche l l and Chalfant 1984). S i x t h , because of heterogeneous d i s t r i b u t i o n of OBs i n time and space, more ind iv id u a ls may escape i n f e c t i o n in nature than in my experiments. Hence, t h i s would reduce the rate and magnitude of res is tance to TnSNPV in f i e l d populat ions of cabbage loopers . The magnitude and ra te of evo lu t ion of TnSNPV may a lso d i f f e r in the f i e l d . F i r s t , the v i ru lence of TnSNPV may be more v a r i a b l e i n the w i ld than i t was i n my o r i g i n a l v i r a l s tocks . Second, in my experiments, I d id not consider t ransmission of the disease wi th in an insect generat ion. A l l 181 v i r a l t ransmission occurred across generat ions. Transmission of the disease wi th in a generation w i l l favour increased v i ru lence because genotypes that k i l l the host qu ick ly w i l l produce more secondary in fec t ions and w i l l have higher i n t r i n s i c reproductive rates (Equation 3 .1) . T h i r d , i f many insects escape in fec t ions in nature by chance, then res is tance w i l l evolve more slowly and the v i r u s w i l l a lso evolve more s lowly. It should a lso be poss ib le to extrapolate these f ind ings to other species of i n s e c t s . For example, s u s c e p t i b i l i t y to NPVs and other baculoviruses has already been shown to vary among populat ions in severa l species and to be g e n e t i c a l l y determined. As with cabbage loopers , the magnitude and rate of coevolut ion i n these species i s l i k e l y to depend on the b i o t i c and a b i o t i c fac tors out l ined in the previous two paragraphes. Impl icat ions for the use of engineered v i ruses A great deal of t ime, e f f o r t and c a p i t a l are present ly being invested into engineering NPVs by e i ther i n s e r t i n g genes coding for insect toxins or removing the egt gene from the genome of NPVs. If t h i s genetic manipulation only reduces time to k i l l , then i t w i l l not provide a stronger s e l e c t i v e 182 pressure and I would expect that insects w i l l evolve res is tance to engineered v i ruses to a comparable l e v e l and at a s i m i l a r rate than non-manipulated NPVs. However, i f at the same dose, engineered v i ruses k i l l more insects ( i . e . they are more v i r u l e n t ) , I p red ic t that insec ts w i l l evolve f a s t e r and greater res is tance to engineered v i ruses than to non-manipulated ones. In a d d i t i o n , the increased v i ru lence conferred through genet ic engineering could i n d i r e c t l y cause the ex t inc t ion of the v i r u s that i s present ly i n nature. That i s because the wi ld type v i r u s would not be s u f f i c i e n t l y v i r u l e n t to k i l l the host and would eventual ly be inac t iva ted by u l t r a v i o l e t l i g h t . Thus, the pest could p o ss ib ly a t t a i n higher l eve ls fo l lowing the re lease of engineered v i r u s e s . App l ica t ions s t ra teg ies for NPV i n s e c t i c i d e s There are two broad s t ra teg ies of a p p l i c a t i o n for NPV i n s e c t i c i d e s (Podgwaite 1985). F i r s t , they can be used l i k e chemicals and appl ied repeatedly to ensure shor t -term cont ro l of the pest i n s e c t . Second, the c l a s s i c a l b i o l o g i c a l approach cons is ts of doing one or a few int roduct ions and for the v i r u s to form a c lose a s s o c i a t i o n with the target insect and provide long-term cont ro l of the 183 pest . Because a g r i c u l t u r a l systems are f requent ly d isturbed and ephemeral, the v i r u s may not have enough time to e s t a b l i s h i t s e l f i n such hab i ta ts . Hence, i n agroecosystems, NPVs may provide bet ter pro tec t ion against herbivores when used l i k e chemicals. Here, I suggest that NPVs be appl ied when the insect i s young because they are are most suscept ib l to NPVs and there w i l l be less damage to crops . In a d d i t i o n , at l eas t for T. ni. , insects that survive the disease when in fec ted at a young age may have impaired reproductive success and t h i s could have fur ther adverse e f f e c t s on the populat ion dynamics of the host . Conversely, s i l v i c u l t u r a l systems are more s table and l o n g - l i v e d and may be provide a bet ter opportunity for the v i rus to e s t a b l i s h i t s e l f . In a d d i t i o n , the greater complexity of the habi tat may provide more refuges for the v i rus to escape i n a c t i v a t i o n by u l t r a v i o l e t l i g h t and al lowing i t to p e r s i s t for longer per iods of t ime. Trees may a lso be able to susta in more damage from herbivores without i t causing appreciable economic l o s s e s . The r e s u l t of my experiments suggest that even i f insects evolve 25 f o l d res is tance to the NPVs, the v i r u s may s t i l l be able to k i l l about ha l f of the pest populat ion. However, I am unable to 184 determine i f t h i s i s an acceptable l e v e l of p ro tec t ion for t r e e s . Future studies The r e s u l t s of my experiments have generated many add i t iona l questions for future s tud ies : (1) What i s the h e r i t a b i l i t y of res is tance of cabbage loopers to TnSNPV in the f i e l d ? (2) What i s the mechanism of res is tance of cabbage loopers to the TnSNPV? (3) How many genes cont ro l the res is tance of cabbage loopers to TnSNPV? (4) Are there many TnSNPV genotypes? And, does v i ru lence vary across these i s o l a t e s ? (5 ) How would cabbage loopers and TnSNPV coevolve i f a s i n g l e or a mixture v i r a l i s o l a t e ( s ) was (were) used? (6) Is res is tance to TnSNPV more l i k e l y to evolve in some parts of the cabbage looper range than others? (7) What i s the e f f e c t of t ransmission of the disease wi th in a generation of T. n i on cabbage looper - TnSNPV coevolution? 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Sarma [eds] , Prote in engineering: a p p l i c a t i o n i n sc ience , medecine and industry . Academic Press Inc . , Orlando, (I) Wiygul , G. and P. P. Sikorowski . 1978. Oxygen uptake i n tobacco budworm larvae (He l io th is v i rescens) in fec ted with cytoplasmic polyhedrosis v i r u s . J . Invert . Patho l . 32:191-195. (5) Wiygul, G. and P. P. Sikorowski . 1991. Oxygen uptake in l a r v a l bollworms (He l io th is zea) in fec ted with i r i d e s c e n t v i r u s . J . Invert . Patho l . 58:252-256. (5) Woods, S. A. and J . S. E l k i n t o n . 1987. Bimodal patterns of mor ta l i ty from nuclear polyhedrosis v i r u s in gypsy moth (Lymantria d ispar) populat ions. J . Invert . Pa tho l . 50:151-224 157. (I) Young, S. Y. and W. C. Year ian . 1990. Transmission of nuclear polyhedrosis v i r u s by the p a r a s i t o i d M i c r o p l i t i s croceipes (Hymenoptera: Braconidae) to H e l i o t h i s v i rescens (Lepidoptera: Noctuidae) on soybean. Envi ron. Entomol. 19:251-256. (I) Zar , J . H. 1987. B i o s t a t i s t i c a l a n a l y s i s . P r e n t i c e - H a l l , Inc, Englewood C l i f f s . (5) Zelazny, B . , A. R. A l f i l e r and A. Lolong. 1989. P o s s i b i l i t y of res is tance to a baculovi rus in a populat ion of coconut rhinoceros beet le (Oryctes rh inoceros) . FAO Plant Prot . B u l l . 37:77-82. (1,3) Zelazny, B . , A. Lolong and A . M. Crawford. 1990. Introduct ion and f i e l d comparison of baculov i rus s t r a i n s against Oryctes rhinoceros (Coleoptera: Scarabaeidae) i n the Maldives. Envi ron. Entomol. 19:1115-1121. (3) Zelazny, B . , A. Lolong and B. Pattang. 1992. Oryctes rhinoceros (Coleoptera: Scarabaeidae) populat ions suppressed by a bacu lov i rus . J . Invert . Pa tho l . 59:61-68. (I) 225 APPENDIX ONE The E f f e c t of Ingestion Time of Treated Plugs on the S u s c e p t i b i l i t y of Trichoplusia n i to the Singly-Embedded Nuclear Polyhedrosis Virus of T_;_ n i . A va r i e ty of techniques have been used to administer baculoviruses to larvae (Entwist le and Evans 1985). The two most widely used methods are the d ie t contamination and droplet feeding techniques. The d i e t contamination technique (the inocu la t ion techniques I used i n my experiments) cons is ts of present ing a small plug of a r t i f i c i a l d i e t or leaf d i s c inoculated with v i r u s to larvae and usua l ly al lowing them 24 hr to consume the t reated d i e t or d i s c . The droplet feeding technique cons is ts of al lowing larvae 1 hr or l ess to imbibe a small droplet of v i r u s and dye s o l u t i o n (Hughes and Wood 1981). The drople t feeding technique i s be l ieved by some to be super ior predominantly because larvae are in fected more synchronously. With the d i e t contamination procedure, the a c q u i s i t i o n time of the dose i s dependent on l a r v a l feeding 226 rate and may vary considerably between i n d i v i d u a l s . Larger and more vigorous individuals may feed f a s t e r and have a lower p r o b a b i l i t y of s u r v i v a l than smaller larvae that ingest the treated d i e t over a longer period. This asynchrony i n inoculation time could a f f e c t the accuracy and r e p e a t a b i l i t y of bioassays and mask sublethal e f f e c t of baculoviruses (Sait et a l . 1994a). In t h i s study, I inoculated cabbage loopers (Trichoplusia ni) with the singly-embedded nuclear polyhedrosis virus of T. n i (TnSNPV) using the d i e t contamination technique and examined whether the time to consume the plug influenced s u r v i v a l . I reared T. n i larvae i n d i v i d u a l l y from hatch in 25 ml p l a s t i c cups as described i n Chapter 1. At 120 or 168 hr post-hatch, I transferred the larvae to a second cup containing only a plug of d i e t (thickness = 2-3 mm, diameter = 5 mm except i n T r i a l 3-120 hr thickness = 1-2 mm, diameter = 5 mm see below). I defined time zero as the time c a t e r p i l l a r s were transferred to the cup containing the plug. Larvae were checked every 3 hr and returned to t h e i r o r i g i n a l cup i f they had completely consumed the plug. I subsequently checked the larvae d a i l y . I conducted t h i s experiment three times for each i n f e c t i o n age. I also weighed the larvae p r i o r 227 to i n f e c t i o n i n T r i a l 1-120 hr and T r i a l s 1-and 2-168 hr. Diagnosis of mortality due to TnSNPV was based on gross pathology. A l l the individuals that were treated with TnSNPV and that died exhibited the t y p i c a l signs of nucleopolyhedrosis. None of the control larvae died during any of these experiments. Within a given t r i a l , the majority of larvae f i n i s h e d eating the plug within 12 hr of one another. For the 120 hr group, 71% f i n i s h e d the plug between 21-33 hr p.t. (hr p.t. = hr post-transfer to the treated plug) in T r i a l 1, 75% between 33-45 hr p.t. i n T r i a l 2 and 81% f i n i s h e d between 9-18 p.t. i n T r i a l 3. For the 168 hr group, 73% finished between 3-15 hr p.t. i n T r i a l 1, 69% between 3-15 hr p.t. i n T r i a l 2 and 69% between 6-18 hr p.t. i n T r i a l 3. The modal plug consumption time of surviving males and females and larvae succumbing to TnSNPV are given Table A.1. I used stepwise multiple l o g i s t i c regression to investigate the e f f e c t of TnSNPV dose, l a r v a l weight at i n f e c t i o n and time to consume the plug on s u r v i v a l . Virus dose always entered the model on the f i r s t step (standardized l o g i s t i c regression c o e f f i c i e n t = -0.81 to -0.171, a l l P's < 0.001). I could not analyze the e f f e c t of dose i n T r i a l 2 -120 hr because a l l larvae were treated with 750 OBs. 228 Larval weight at i n f e c t i o n was s i g n i f i c a n t i n T r i a l 1-168 hr; heavier larvae had a greater p r o b a b i l i t y of s u r v i v a l (standardized l o g i s t i c regression c o e f f i c i e n t s = 0.41, P = 0.007). Plug consumption time was not related to survival i n any of the 6 t r i a l s ( a l l P's > 0.50). Two other l i n e s of evidence suggest that the time to consume plugs does not influence s u s c e p t i b i l i t y to v i r u s . (1) I shortened l a r v a l consumption time by using plugs that were half as thick i n T r i a l 3-120 hr but sur v i v a l at 750 TnSNPV OBs was 59% and nearly i d e n t i c a l to T r i a l s 1- (59%) and 2-120 hr (56%; Table 1). (2) Although modal plug consumption times varied among t r i a l s i n the 168 hr experiments, the survival of larvae treated with 1500 OBs was comparable i n a l l 3 t r i a l s ( T r i a l 1 = 65%, T r i a l 2 = 57%, T r i a l 3 = 64%; Table A . l ) . A major c r i t i c i s m of the d i e t contamination technique has been that larvae may be infected asynchronously and that t h i s could influence the s u r v i v a l of indivi d u a l s treated with NPVs. The res u l t s of t h i s study suggest that t h i s concern i s not warranted. 229 o o o LO CO o : co o o o IT) o ^ Si-CM m O CO 03 O Q > Q. Z CO c o o in r--C-CM 0 5 CD 0-CD" o o in 05 oT" ' CO CT) CO i T ' ^ ' " CM 1 0 3 ^ a CM o co T - co T - t-~ CM CM CM CO CM CO IO O) s CO CM ^ 00 CO CD in CM T -CO o~ in ™£! CM" CM"!£ ^ o in" CM CD CD 00 CO : u. x CO I— CO < (0 o CM 00 CD 

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