Bacillus thuringiensis - ACS Publications - American Chemical

20 δ-Endotoxin of Bacillus thuringiensis var. israelensis Broad-Spectrum Toxicity and Neural Response Elicited in Mice and Insects R. MICHAEL A. RANDALL

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ROE , PETER Y. K. CHEUNG , ALFORD

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BRUCE D. HAMMOCK ,

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DAN BUSTER , and

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Downloaded by UNIV OF LIVERPOOL on May 29, 2018 | https://pubs.acs.org Publication Date: April 26, 1985 | doi: 10.1021/bk-1985-0276.ch020

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Department of Entomology, North Carolina State University, Raleigh, NC 27695-7613 Department of Entomology, University of California, Davis, CA 95616 Department of Entomology, University of Maine, Orono, ME 04469

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The alkaline-dissolved Bacillus thuringiensis israelensis (BTI) δ-endotoxin when introduced by injection was biologically active against a wide spectrum of host animals including insects from four orders and mice. The LD for dissolved BTI δ-endotoxin in mice was 1.31 PPM and in Trichoplusia ni (Lepidoptera: Noctuidae) 3.71 PPM. Neuromuscular effects like heart cessation, lost coordination, tremor, and paralysis were observed in test animals. Using the appearance of lactate dehydrogenase in insect hemolymph post-injection as a cytosolic marker, we found that dissolved BTI δ-endotoxin was cytotoxic. In vivo recordings of activity in the ventral nerve cord post-injection indicated that dissolved BTI δ-endotoxin at the T. ni LD elicited hyperexcitability and then nerve death as was also the case for the organophosphate, methamidophos. The cytotoxin phospholipase-A when injected at its LD elicited no neural response. BTI poisoning was also temperature dependent while BTI cytotoxicity was not. Proteins at 24, 27, 35, 49 and 68K daltons were resolved from the dissolved BTI δ-endotoxin. These were introduced in various combinations by injection and ingestion into mice and insects and compared to the alkaline-dissolved Bacillus thuringiensis kurstaki δ-endotoxin. 50

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W i t h i n t h e sporangium o f t h e b a c t e r i u m B a c i l l u s t h u r i n g i e n s i s (BT) i s s y n t h e s i z e d a p a r a s p o r a l , p r o t e i n a c e o u s c r y s t a l ( 1 - 2 ) t h a t has found w i d e s p r e a d use as a b i o l o g i c a l c o n t r o l agent ( 3 ) . T h i s c r y s t a l i s commonly r e f e r r e d t o as t h e "δ-endotoxin" as s u g g e s t e d by Heimpel ( 4 ) . The taxonomy o f BT i s based on t h e s e r o l o g y o f t h e f l a g e l l a r Η a n t i g e n 0 5 ) , and 29 s u b s p e c i e s and 26 s e r o t y p e s have been i d e n t i f i e d (6). The δ-endotoxins from t h e

0097-6156/85/0276-0279$06.00/0 © 1985 American Chemical Society Hedin et al.; Bioregulators for Pest Control ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

BIOREGULATORS FOR


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m a j o r i t y o f the s e r o t y p e s a r e t o x i c when i n g e s t e d by more than 182 s p e c i e s o f i n s e c t s , p a r t i c u l a r l y i n the e c o n o m i c a l l y i m p o r t a n t o r d e r , L e p i d o p t e r a (3). The m a j o r i t y o f the r e s e a r c h has c e n t e r e d on B a c i l l u s t h u r i n g i e n s i s s u b s p e c i e s k u r s t a k i (BTK) because o f i t s l a r v i c i d a l a c t i v i t y a g a i n s t major a g r i c u l t u r a l p e s t s i n the o r d e r L e p i d o p t e r a . The s e r o t y p e H14, B a c i l l u s t h u r i n g i e n s i s s u b s p e c i e s i s r a e l e n s i s ( B T I ) , d i f f e r s from the o t h e r s e r o t y p e s , however, by b e i n g h i g h l y t o x i c to members o f the i n s e c t o r d e r D i p t e r a (7-8) and y e t has l i t t l e known t o x i c i t y t o Lepidoptera. The p r o s p e c t o f employing BTI t o c o n t r o l m o s q u i t o e s , b l a c k f l i e s , or o t h e r m e d i c a l l y important i n s e c t p e s t s has s t i m u l a t e d g r e a t i n t e r e s t i n e l u c i d a t i n g the m o l e c u l a r b a s i s f o r i t s mode o f a c t i o n (_3). A l l o f the BT δ-endotoxins are a l s o o f s p e c i a l i n t e r e s t because they appear to be h i g h l y s e l e c t i v e a g a i n s t i n s e c t s and seem t o pose no h e a l t h r i s k s to humans or livestock. The δ-endotoxin o f BTK upon i n g e s t i o n by l a r v a l L e p i d o p t e r a i s q u i c k l y a c t i v a t e d by h i g h gut pH and gut p r o t e o l y t i c a c t i v i t y ( 9 - 1 1 ) ; gut e p i t h e l i a l c e l l s s w e l l , v a c u o l e s form, and then the c e l l s s e p a r a t e from the basement membrane and each o t h e r u l t i m a t e l y d i s r u p t i n g the gut-hemocoel b a r r i e r (12-15). Similar o b s e r v a t i o n s i n mosquito l a r v a e f e d BTI (1_6) l e d to the g e n e r a l a c c e p t a n c e t h a t the BT i n s e c t i c i d a l a c t i v i t y was d i r e c t e d a g a i n s t , i f not r e s t r i c t e d to the gut e p i t h e l i u m o f the h o s t (12-13). An i n i t i a l symptom o f BTK p o i s o n i n g i s gut p a r a l y s i s (17) and i t was h y p o t h e s i z e d t h a t an i n c r e a s e i n the hemolymph pH from the leakage o f a l k a l i n e gut c o n t e n t s (17) or the i n f l u x o f K i n t o the hemocoel caused t h i s p a r a l y s i s ( 1 8 - 1 9 ) . These s t u d i e s l e d to the d i s c o v e r y t h a t BTK d i g e s t s a p p l i e d to the v e n t r a l nerve c o r d o f the c o c k r o a c h , P e r i p l a n e t a americana ( O r t h o p t e r a : B l a t t i d a e ) caused e x c i t a t i o n and then nerve b l o c k a g e (20-21 ) which appeared to be p r e s y n a p t i c i n o r i g i n (20). Other s t u d i e s have shown t h a t the a l k a l i n e - d i s s o l v e d BTI δ-endotoxin i s c y t o t o x i c to a number o f d i f f e r e n t c e l l l i n e s from i n s e c t s and mammals (3,22-24) and has a h i g h a f f i n i t y f o r s p e c i f i c p h o s p h o l i p i d s i n the plasma membrane (25). Thus, the o b j e c t i v e o f t h i s s t u d y i s to a s s e s s the t o x i c i t y o f a l k a l i n e - d i s s o l v e d BTI i n t r o d u c e d i n t o i n s e c t s and mice by f e e d i n g and i n j e c t i o n and t o a s s e s s the r o l e o f c y t o t o x i c i t y and n e u r o t o x i c i t y i n m o r t a l i t y when d i s s o l v e d δ-endotoxin i s i n j e c t e d i n t o i n s e c t s . +

SDS-PAGE A n a l y s i s

o f BTK

and

BTI

δ-Endotoxin

BTK and BTI s t r a i n IFC-1 were p r o v i d e d by Biochem P r o d u c t s - US D i v i s i o n ( S a l s b u r y Labs., I n c . ) . BTI was a l s o i s o l a t e d from a commercial p r e p a r a t i o n p r o v i d e d by Sandoz Inc., c u l t u r e d on GYS medium ( 2 6 ) . BTK and BTI t o x i n was p r e p a r e d i n an analogous manner. Spores and c r y s t a l s were s e p a r a t e d from c e l l d e b r i s by r e p e a t e d washing w i t h water and c e n t r i f u g a t i o n . BTI c r y s t a l s were s u b s e q u e n t l y s e p a r a t e d from spores by R e n o g r a f i n d e n s i t y g r a d i e n t c e n t r i f u g a t i o n (27_) and BTK c r y s t a l s by d i s c o n t i n u o u s sucrose gradient c e n t r i f u g a t i o n Ο ) . C r y s t a l s were then d i s s o l v e d by i n c u b a t i o n f o r 3 h i n 0.5% Na2C03 (pH 11.0) and d i a l y z e d i n t o 0.025 M sodium phosphate (pH 8.0) f o r s t o r a g e a t

Hedin et al.; Bioregulators for Pest Control ACS Symposium Series; American Chemical Society: Washington, DC, 1985.


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Effects of δ-Endotoxin in Mice and Insects

-60°C. BTI (Sandoz) was further p u r i f i e d by DEAE-Cellulose (DE-52, Whatman, 30 ml) i n 0.025 M Tris-HCL (pH 8.00), eluted with a 10 h, 100 ml, 0.0-0.5 M NaCl linear gradient; by acid p r e c i p i t a t i o n where the DEAE elutant was dialyzed into 0.05 M sodium acetate (pH 4.5) and the percipitate removed by centrifugation; and by Sephadex G-75 super fine (Pharmacia) gel permeation chromatography (95 χ 1.3 cm i . d . column) i n 0.025 M sodium phosphate (pH 8.0). Alkaline-dissolved and p a r t i a l l y p u r i f i e d δ-endotoxin was analyzed by 12.5% SDS-PAGE (2j8), stained with Coomassie b r i l l i a n t blue (Figure 1). Incubation of BTK and BTI crystals i n 0.5% Na2C03 (pH 11.0) s o l u b i l i z e d a number of protein components (Figure 1). For BTK, there were a number of proteins at 64K daltons and higher (Track 1). The standard procedure of washing BTK crystals with 1 M NaCl before s o l u b i l i z a t i o n removes endogenous proteinases and results in an enriched 130K dalton protein as the predominant component. This procedure had l i t t l e effect on the immunoreactivity of the s o l u b i l i z e d c r y s t a l or i t s t o x i c i t y i n our studies. For BTI (Sandoz, Track 2) there were proteins at 24, 27, 35, 49 and 68K daltons. Of the lower molecular weight Sandoz BTI components, the 27K proteins were the prédominent component i n the Salsbury BTI (Track 8). Differences i n the protein p r o f i l e of the ό-endotoxin from different BT v a r i e t i e s have been reported previously (11,30-31). A l l a l k a l i n e - s o l u b i l i z e d 6-endotoxin of BTI (Sandoz) adsorbed to DE-52 and eluted i n one peak (Track 3) with an apparent concentration of the 68K component. The acid precipitate (Track 4) was enriched with the 35K component which was r e - s o l u b i l i z e d only at high pH. Because of i t s limited s o l u b i l i t y , the acid precipitate could not be bioassayed i n later studies. The soluble f r a c t i o n was enriched with the 24K and 27Κ· components (Track 5). Gel permeation chromatography enriched the 27 and 24K proteins (Tracks 6 and 7, r e s p e c t i v e l y ) . 6-Endotoxin Toxicity i n Mice and Insects BTI and BTK alkaline-dissolved and p a r t i a l l y p u r i f i e d δ-endotoxins were injected and/or fed to insects of 6 orders and to mice (Tables I and I I ) . The δ-endotoxin was injected i n 0.15 M NaCl, 0.05 M Na2HP04, and 0.02 M K H 2 P O 4 at pH 7.2 into the insect hemocoel or intraperitoneally into mice. In feeding experiments, the toxin was dissolved i n 5% sucrose and force-fed in 2 μΐ volumes, to Trichoplusia n i and Heliothis zea (Lepidoptera: Noctuidae). Aedes aegypti (Diptera: Culicidae) larvae fed for a standard incubation period i n water containing BT toxin preparations; adults were given r e c t a l injections (32). Mice were also given BT by gavage. Data collected were subjected to Probit analysis (33). The alkaline-dissolved 6-endotoxin of BTI (Sandoz, Track 2) was toxic by i n j e c t i o n to a l l animals tested except Tenebrio molitor (Coleoptera: Tenebrionidae) (Table I ) . Mice, Trichoplusia n i , and Periplaneta americana were the most sensitive, the L D 5 0 being 1.3, 3.7 and 4.4 PPM, respectively. The s u s c e p t i b i l i t y to BTI poisoning also varied s i g n i f i c a n t l y within a single insect family (the Noctuidae) with the L D 5 0


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F i g u r e 1. SDS-PAGE a n a l y s i s o f a l k a l i n e - d i s s o l v e d B a c i l l u s t h u r i n g i e n s i s s u b s p e c i e s k u r s t a k i (BTK) and i s r a e l e n s i s ( B T I ) δ-endotoxin a t 25 y g per t r a c k : ( 1 ) BTK δ-endotoxin from Biochem P r o d u c t s - US D i v i s i o n ( S a l s b u r y Labs., I n c . ) , (2) BTI δ-endotoxin from Sandoz I n c . , (3) BTI (Sandoz) δ-endotoxin p u r i f i e d by DEAE-anion exchange chromatography, ( 4 ) p e r c i p i t a t e formed a f t e r d i a l y s i s o f BTI (Sandoz) δ-endotoxin i n t o pH 4.5 sodium a c e t a t e b u f f e r , (5) s o l u b l e f r a c t i o n a f t e r d i a l y s i s o f BTI (Sandoz) δ-endotoxin i n t o pH 4.5 sodium a c e t a t e b u f f e r , (6) BTI (Sandoz) δ-endotoxin p u r i f i e d by Sephadex G-75 g e l f i l t r a t i o n chromatography a t Rf 1.35, (7) a t Rf 1.58, and (8) BTI s t r a i n IFC-1 δ-endotoxin from Biochem P r o d u c t s - US D i v i s i o n ( S a l s b u r y Labs., I n c . ) . S, m o l e c u l a r w e i g h t s as i n d i c a t e d X1000 f o r b o v i n e serum a l b u m i n (BSA), o v a l b u m i n (OA), t r y p s i n , and m y o g l o b i n . Reproduced w i t h p e r m i s s i o n from R e f . 29. C o p y r i g h t 1984, Academic P r e s s , I n c .


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T a b l e I . I n j e c t e d t o x i c i t y o f a l k a l i n e - s o l u b i l i z e d BTI (Sandoz, T r a c k 2) and BTK ( S a l s b u r y , T r a c k 1) δ-endotoxin.


Animal - A or L* Aedes a e g y p t i - A (Diptera: Culicidae) Musca domesticus - A ( D i p t e r a : Muscidae) Trichoplusia n i - L (Lepidoptera: Noctuidae) H e l i o t h i s zea - L (Lepidoptera: Noctuidae) Tenebrio m o l i t o r - L (Coleoptera: Tenebrionidae) Oncopeltus f a s c i a t u s - A (Hemiptera: L y g a e i d a e ) P e r i p l a n e t a americana - A (Orthoptera: B l a t t i d a e ) Swiss-Webster Mice

*Adult +

24h BTI L D + 5 0

24h BTK L D + 5 0

11.6 + 2.2 (3.16) 10.9 + 2.2 (2.17) 3.71 + 0.32 (3.22) 73.6 + 3.0 (19.23) >100

>900

27.7 + 7.0 (1.96) 4.42 + 0.36 (7.04) 1.31 + 0.23 (4.47)

>300

>150 >130 >100 >100

>20 >30

or Larva.

PPM o r mg/kg body weight + 1 S.D. w i t h analysis i n parenthesis.

slope of probit

r a n g i n g from 3.7 PPM f o r T. n i t o 73.6 PPM f o r H e l i o t h i s z e a . The b a s i s f o r t h i s d i f f e r e n c e i s unknown but these s p e c i e s d i f f e r e n c e s c o u l d be u s e f u l i n the e l u c i d a t i o n o f the mechanism for t o x i c i t y . BTI t o x i c i t y by i n j e c t i o n a l s o was n o t p e c u l i a r t o the Sandoz s t r a i n but was a l s o noted f o r the IFC-1 s t r a i n from S a l s b u r y ( T a b l e I I I ) . By c o n t r a s t the a l k a l i n e - d i s s o l v e d δ-endotoxin o f BTK ( T r a c k 1) showed no t o x i c i t y when i n j e c t e d i n t o the same s p e c i e s ( T a b l e I ) . Obvious fundamental d i f f e r e n c e s e x i s t between t h e d i s s o l v e d δ-endotoxin o f BTI and BTK. When the p u r i f i e d , p a r a s p o r a l c r y s t a l o f BTI (Sandoz) was f e d t o A. a e g y p t i l a r v a e , t h e L C 5 0 was 2.95 + 0.59 ng/ml. A l k a l i n e - s o l u b i l i z a t i o n d e c r e a s e d the t o x i c i t y s i g n i f i c a n t l y t o an L C 5 0 o f 2.29 + 0.06 yg/ml and r e c t a l i n j e c t i o n s i n a d u l t s produced a L D 5 0 o f 54.5 + 3.1 PPM ( T a b l e I I ) . These f i n d i n g s were c o n s i s t e n t w i t h p r e v i o u s work ( 3 4 ) . D i s s o l v e d BTI δ-endotoxin when f e d t o L e p i d o p t e r a and mice as e x p e c t e d ( 3 ) was not t o x i c ( T a b l e I I ) . The BTI IFC-1 s t r a i n was a l s o s i m i l a r t o the Sandoz s t r a i n i n t h a t b o t h were t o x i c when f e d t o A. a e g y p t i ( T a b l e I I I ) . BTK d i s s o l v e d δ-endotoxin when g i v e n o r a l l y was t o x i c o n l y t o t h e l e p i d o p t e r a n , T. n i ( T a b l e I I ) . The r e s u l t s from a l l o f t h e s e f e e d i n g experiments were c o n s i s t e n t w i t h p r e v i o u s r e p o r t s t h a t BTK when f e d t o l e p i d o p t e r a n s i s a c t i v e w h i l e BTI i s t o x i c t o o n l y c e r t a i n d i p t e r a n s ( 3 ) and i s s u p p o r t i v e e v i d e n c e t h a t the BTI and BTK p r e p a r a t i o n s used i n our s t u d i e s were s i m i l a r t o p r e p a r a t i o n s p r e v i o u s l y used by o t h e r investigators. F u r t h e r m o r e , c r o s s - c o n t a m i n a t i o n between BTI and



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BTK as determined by ELISA was less than 0.01%, the detectable l i m i t of the assay (35). The combined 24, 27, and 35K components of BTI (Sandoz) δ-endotoxin (Figure 1, Tracks 2 and 3) had an equivalent t o x i c i t y

Table I I . Oral t o x i c i t y of a l k a l i n e - s o l u b i l i z e d BTI (Sandoz, Track 2) and BTK (Salsbury, Track 1) δ-endotoxin.


Animal A. aegypti Larva Adult (ENEMA) T. n i Larva H. zea Larva Swiss-Webster Mice

24h BTI LD +

24h BTK

2.29 + 0.06* (12.41) 54.5 + 3.1 (6.37) >50

>40*

50

>35

LD50+

>900 2.30 + 0.27 (2.85) >35

>30

>30

+

PPM or mg/kg body weight + 1 S.D. with slope of probit analysis i n parenthesis.

*yg/ml of water i n which larvae were incubated.

Table I I I . Toxicity of p a r t i a l l y p u r i f i e d a l k a l i n e s o l u b i l i z e d BTI δ-endotoxin fed and injected into A. aegypti and T. n i , respectively. A. aegypti Toxin Alkaline dissolved, Sandoz (Track 2) DEAE (Track 3) pH 4.5 soluble (Track 5, 27 & 24K) G-75, Rf 1.35 (Track 6, 27K) G-75, Rf 1.53 (Track 7, 24K) Alkaline dissolved, Salsbury (Track 8, 27K)

24h

LC50*

2.29 + 0.06 (12.4) 1.91 + 0.30 (2.92) 2.02 + 0.54 (2.06) 2.71 + 0.12 (5.72) >20 5.78 + 0.42 (4.76)

T. n i 24h

LD50+

3.71 + 0.32 (3.22) 1.96 + 0.64 (2.16) 1.95 + 0.14 (4.32) 3.54 + 0.50 (3.28) 2.97 + 0.18 (7.00) 6.09 + 0.46 (4.67)

*yg/ml +1 S.D. with slope of probit analysis i n parenthesis. Larvae were incubated i n water with BTI δ-endotoxin added. PPM or mg/kg body weight + 1 S.D. with slope of probit analysis i n parenthesis. Larvae were injected.



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before and after DEAE (Table III for both A. aegypti and T. n i ) , even though there appeared to be a concentration of the 68K com ponent i n this p u r i f i c a t i o n step. The 24 and 27K component i n d i v i d u a l l y (Figure 1, Tracks 7 and 6, respectively) also had an equivalent t o x i c i t y when injected into T. n i (Table III) but the 24K component (Figure 1, Track 7) was not toxic when fed to A. aegypti whereas the 27K component was toxic (Table I I I ) . This i n a c t i v i t y cannot be explained by the absence of the 35 and 68K components i n Track 7 (Figure 1) because these same components are also absent i n Track 5 and yet Track 5 retained oral t o x i c i t y to A. aegypti (Table I I I ) . In fact the absence of the 68 and 35K components (Figure 1, Tracks 5 and 7) likewise did not affect the T. n i a c t i v i t y (Table I I I ) . Thus i t appears that at least the 27K proteins are necessary for A. aegypti oral t o x i c i t y while both the 24 and 27K components can impart t o x i c i t y to T. n i when injected. The 27K component was also toxic i n both A. aegypti and T. n i regardless of the source (Track 6 and Track 8, Figure 1 and Table I I I ) . Neural Toxicity of BTI 6-Endotoxin The i n j e c t i o n of alkaline-dissolved BTI 6-endotoxin led to a number of immediate neuromuscular effects (Table IV) including,

Table IV. Symptoms e l i c i t e d after the i n j e c t i o n of a l k a l i n e dissolved BTI δ-endotoxin (Track 2) into mice and insects Trichoplusia ni (5 PPM)

Periplaneta americana (6 PPM)

Swiss-Webster Mice (1.5 PPM)

0-lh

Mouth palpation of i n j e c t i o n site Increased wandering Heart arrest Abdominal paralysis L i s t side to side when crawling Total paralysis & flaccidity

Loss of motor activity

Ruffled fur Lost alertness Not i n q u i s i t i v e Reduced responsiveness Slow i n righting themselves Breathing shallow Lost a c t i v i t y in hind legs

20-24h

Localized black ening of the body Total blackening of the body No response to head stimulation

In Survivors Failure to right themselves Tremor

In Survivors, Constipation Dead animals with a pinched waist

Time PostInjection



286


i n the insects tested, l i s t i n g from side to side when crawling, heart arrest, paralysis, and tremors. These symptoms were observed for both the Sandoz and Salsbury BTI 6-endotoxin and were also observed for p a r t i a l l y p u r i f i e d BTI δ-endotoxin (Table III). The symptoms observed i n mice were somewhat similar to those observed i n botulism poisoning (a neurotoxin), which included a loss of alertness, shallow breathing, and i n some cases lost a c t i v i t y i n the hind legs. Dead mice had a pinched waist, a sign of diaphram arrest. The symptoms observed i n insects following the i n j e c t i o n of BTI 6-endotoxin were c l e a r l y d i f f e r e n t from those following the ingestion of BTK 6-endotoxin. When T. n i were fed BTK δ-endotoxin, there was regurgitation within 15 min, a t o t a l cessation of feeding u n t i l death, and no overt neuromuscular anomalies. The i n j e c t i o n of a l k a l i n e dissolved BTK 6-endotoxin produced no obvious adverse effects i n insects or mice. A number of other lines of evidence also suggested that there may be another mode-of-action for BTI poisoning by i n j e c t i o n other than i t s known, general c y t o l y t i c a c t i v i t y (3,22-24). Using the appearance of cytosolic lactate dehydrogenase (LDH) i n insect hemolymph post-injection as a marker for c y t o t o x i c i t y (36-37), we found that dissolved BTI δ-endotoxin was a potent cytotoxin. When T. n i , however, were injected with dissolved BTI 6-endotoxin and then incubated at 28, 15, and 9°, there was an increase i n the L D 5 0 with a decrease i n temperature (Figure 2) but the LDH levels at 3.5 PPM BTI were unaffected by temperature. A pharmocological study of ventral nerve cord function i n T. n i also suggested that alkaline-dissolved BTI 6-endotoxin was a f f e c t i n g the insect nervous system as a nerve poison (Figures 3 and 4 ) . After 7-60 min post-injection of alkaline-dissolved BTI δ-endotoxin at the L D 5 0 concentration of 3.7 PPM, the ventral nerve cord of T. n i exhibited spontaneous-high frequency discharges. This was followed by a reduced baseline a c t i v i t y and s e n s i t i v i t y to sensory stimulation (S, Figure 4) at 24 h post-treatment. By 2-90 min post-injection of methamidophos, there also was spontaneous-high frequency discharge which lasted 20 min - 6 h and was followed by a reduced baseline a c t i v i t y and s e n s i t i v i t y to sensory stimulation (S, Figure 4) by 24 h. The response to methamidophos was s l i g h t l y more rapid and sustained than with BTI toxin. Studies also suggested that the primary s i t e of action for BTI might be the peripheral nervous system and that the mode-of-action of BTI and methamidophos on the insect nervous system were probably d i f f e r e n t . When the peripheral nervous system i s severed from the T. n i ventral nerve cord, the methamidophos application s t i l l e l i c i t s spontaneous discharge, but there i s no response to BTI toxin. When the cytotoxin, phospholipase-A2, i s injected into T. n i at i t s L D 5 0 of 35 PPM, there i s no spontaneous-high frequency discharge i n the ventral nerve cord and no reduction i n the stimulus-response (S, Figure 4) as was the case for BTI. A 25K dalton component was isolated at r e l a t i v e l y high purity as determined by SDS-PAGE (Figure 5, Track 3) from alkaline-dissolved BTI δ-endotoxin (Figure 5, Track 1). The


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287



20.

R1

R2

F i g u r e 3. Neurophysiological preparation of T r i c h o p l u s i a n i . Head, t h o r a x and gut a r e removed. Tungsten e l e c t r o d e s were p l a c e d i n t o the hemocoel a l o n g s i d e abdominal g a n g l i o n V I I I ( a t R l ) , the v e n t r a l nerve c o r d ( a t R2) and the abdominal w a l l ( g r o u n d , R£>. I n j e c t i o n s o f a l k a l i n e - d i s s o l v e d BTI δ-endotoxin, methamidophos and p h o s p h o l i p a s e - A 2 were i n t o the second p a i r o f abdominal p r o l e g s ( C h ) . Mechanical sensory s t i m u l a t i o n w i t h a g l a s s probe was a t the a n a l p r o l e g ( S ) . A c t i v i t y i n the v e n t r a l n e r v e c o r d was m o n i t o r e d through 24 h post-treatment (38-40) (see F i g u r e 4 ) .



!

S

s


Sec

0 min

1

24 hr

7-60 min

10 PPM

s

s MMP

0 min

35 PPM

s P-A

j !

2

0 min

24 hr

2-90 min

!

;

s

60 min

j ;

S

24 hr

F i g u r e 4 . Time dependency o f nervous a c t i v i t y i n the v e n t r a l nerve c o r d o f T r i c h o p l u s i a n i i n j e c t e d w i t h 3 . 7 PPM a l k a l i n e d i s s o l v e d BTI δ-endotoxin ( S a n d o z ) , w i t h 1 0 PPM methamidophos (MMP) and w i t h 3 5 PPM p h o s p h o l i p a s e - A 2 ( P - A 2 ) . Mechanical s e n s o r y s t i m u l a t i o n i s g i v e n a t arrow j>. The c o n t r o l r e s p o n s e was the same as t h e r e c o r d i n g f o r P - A 2 . BTI and P-A2 were i n j e c t e d i n t o T. n i a t t h e i r r e s p e c t i v e L D 5 Q .



ROE ET AL.


F i g u r e 5. SDS-Page a n a l y s i s o f a l k a l i n e - d i s s o l v e d Bacillus t h u r i n g i e n s i s i s r a e l e n s i s ( B T I ) δ-endotoxin from Sandoz I n c . a t 25 pg per t r a c k : (1) BTI δ-endotoxin as p r e p a r e d i n F i g u r e 1 ( T r a c k 2 ) , (2) s o l u b l e f r a c t i o n a f t e r d i a l y s i s o f BTI δ-endotoxin i n t o pH 4.5 sodium a c e t a t e b u f f e r , and (3) 25K component from BTI δ-endotoxin a f t e r pH 4.5 p e r c i p i t a t i o n and DEAE-anion exchange chromatography. S, m o l e c u l a r weight markers from t o p t o bottom b o v i n e serum a l b u m i n (68K d a l t o n s ) , ovalbumin (43K), and m y o g l o b i n (16K).



290


s u p e r n a t a n t ( F i g u r e 5, T r a c k 2) a f t e r pH 4.5 p e r c i p i t a t i o n o f t h e crude e n d o t o x i n was f u r t h e r p u r i f i e d by D E A E - C e l l u l o s e (DE-52, Whatman, 30 m l ) i n 0.025 M T r i s - H C l (pH 8.00), e l u t e d w i t h a 48 h, 400 m l , 0.0-0.2 M NaCl l i n e a r g r a d i e n t ( F i g u r e 5, T r a c k 3 ) . The r e s u l t i n g 25K d a l t o n component ( F i g u r e 5, T r a c k 3) a t 0.85 pg/ml produced 50% c e l l l y s i s i n a 1% human r e d b l o o d c e l l s o l u t i o n (25°C., 15 min) and was a l s o h e m o l y t i c a g a i n s t sheep and r a b b i t r e d blood c e l l s . H e m o l y t i c a c t i v i t y i n c r e a s e d f o r the 25K component ( F i g u r e 5, T r a c k 3) a f t e r a 15 s e c i n c u b a t i o n a t 70°C and was i n a c t i v a t e d a t 90°C as was a l s o the case f o r the crude t o x i n ( F i g u r e 5, T r a c k 1 ) . At 25 yg/ml, however, t h e 25K p r o t e i n was n o t t o x i c o r a l l y t o A. a e g y p t i even a f t e r 15 s e c heat t r e a t m e n t s a t 45, 70 o r 90°C n o r was i t l e t h a l when i n j e c t e d i n t o T. n i l a r v a e a t 4.6 PPM ( t h e 48 h L D 5 0 > 4.6 PPM). Injections d i d d i s r u p t l a r v a l - p u p a l metamorphosis l a t e r i n development. The crude t o x i n ( F i g u r e 5, T r a c k 1 ) , however, was t o x i c t o A. a e g y p t i and T. n i a t 45 and 70° b u t n o t a f t e r the 90° t r e a t m e n t . So the 25K component i s c y t o t o x i c b u t does n o t have e i t h e r o r a l t o x i c i t y to A. a e g y p t i o r i n j e c t e d t o x i c i t y t o T. n i . Conclusions I t appears from our s t u d i e s and r e p o r t s i n the l i t e r a t u r e t h a t the p a r a s p o r a l c r y s t a l o f BTI has g u t - t o x i c i t y when f e d t o t h e mosquito ( l j 6 ) , both i n v i t r o and i n v i v o c y t o t o x i c i t y (3,22-24) and i n v i v o n e u r o t o x i c i t y . Injected t o x i c i t y occurred i n a number o f i n s e c t s p e c i e s . The crude a l k a l i n e - d i s s o l v e d δ-endotoxin o f BTI when i n j e c t e d i n t o T. n i was s t r o n g l y c y t o t o x i c and a t i t s L D 5 0 a l s o n e u r o t o x i c . T h i s was u n l i k e the c y t o t o x i n , p h o s p h o l i p a s e - A 2 which demonstrated no n e u r o t o x i c i t y at i t s L D 5 0 . The t o x i c i t y o f BTI ό - e n d o t o x i n i n j e c t e d was a l s o temperature-dependent w h i l e i t s c y t o l y t i c a c t i v i t y was unchanged i n the same temperature r a n g e . A 25K component i s o l a t e d from BTI ( F i g u r e 5, T r a c k 3) was a l s o found t o be c y t o l y t i c b u t when i n j e c t e d had no t o x i c i t y . U n t i l each o f t h e components from the a l k a l i n e - d i s s o l v e d δ-endotoxin o f BTI ( F i g u r e 1, T r a c k 2) can be p u r i f i e d and s e p a r a t e l y t e s t e d f o r c y t o t o x i c i t y and n e u r o t o x i c i t y , the i n t e r r e l a t i o n s h i p s o f t h e s e m o d e s - o f - a c t i o n t o the many p o l y p e p t i d e s found i n the δ-endotoxin o f BTI w i l l be i n q u e s t i o n . The v a r i a t i o n s o b t a i n e d w i t h d i f f e r e n t BTI δ-endotoxin p r e p a r a t i o n s and BTI s t r a i n s w i l l m a g n i f y t h e c o m p l e x i t y o f the problem. N e v e r t h e l e s s , a number o f l i n e s o f e v i d e n c e now e x i s t s t o suggest t h a t i n j e c t e d t o x i c i t y and n e u r o t o x i c i t y a r e not necessary a function of general c y t o l y t i c a c t i v i t y . The e v i d e n c e i s c l e a r t h a t t h e r e a r e a l s o c y t o l y t i c components t h a t demonstrate no gut t o x i c i t y . The f i n d i n g o f s p e c i e s d i f f e r e n c e w i t h i n a s i n g l e f a m i l y o f L e p i d o p t e r a t o the s u s c e p t i b i l i t y o f BTI δ-endotoxin p o i s o n i n g by i n j e c t i o n w i l l be an i m p o r t a n t t o o l f o r studying mode-of-action i n the f u t u r e . Reports on the n e r v e b l o c k i n g a c t i o n from d i g e s t s o f the δ-endotoxin o f BTK ( 2 0 - 2 1 ) , suggest t h a t a n e u r o t o x i c element may be common t o the a c t i o n o f o t h e r members o f the BT complex.


20.

ROE ET AL.

Effects of ^-Endotoxin in Mice and Insects


Acknowledgments Use o f t r a d e names i n t h i s p u b l i c a t i o n does n o t imply endorsement o f the p r o d u c t s named o r c r i t i c i s m o f s i m i l a r ones not mentioned. T h i s paper i s Number 9592 o f the J o u r n a l S e r i e s o f t h e N o r t h C a r o l i n a A g r i c u l t u r e Research S e r v i c e , R a l e i g h , N o r t h C a r o l i n a 27695. R. M. Roe was s u p p o r t e d i n p a r t by t h e Department o f H e a l t h and Human S e r v i c e s , N a t i o n a l S e r v i c e Award 1 F32 GM09223-02 from the N a t i o n a l I n s t i t u t e o f G e n e r a l S c i e n c e s ; and B. D. Hammock by NIEHS Research C a r e e r Development Award 5 K04 ES00107-05. P a r t i a l support f o r t h i s r e s e a r c h was p r o v i d e d by NIEHS Grant ES02710-04, the U n i v e r s i t y o f C a l i f o r n i a G e n e r a l Fund f o r M o s q u i t o R e s e a r c h , and the r e s p e c t i v e s t a t e a g r i c u l t u r a l e x p e r i ment s t a t i o n s . The a s s i s t a n c e o f Dr. C h a r l e s L. Judson and Ms. Mary Ann Montague f o r t h e i r mosquito i n j e c t i o n s o f BTI and BTK i s most a p p r e c i a t e d . A l s o the a s s i s t a n c e o f K e n j i Ota, R a f a e l d e l V e c c h i o , J i m O t t e a , and T e r r y H a n z l i k i n i n s e c t r e a r i n g i s g r a t e f u l l y acknowledged.

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RECEIVED November 15, 1984


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