Development of Photoactivated Compounds as ... - ACS Publications

Chapter 1

Development of Photoactivated Compounds as Pesticides James R. Heitz

Downloaded by LAURENTIAN UNIV on September 8, 2013 | http://pubs.acs.org Publication Date: May 7, 1987 | doi: 10.1021/bk-1987-0339.ch001

Department of Biochemistry, Mississippi Agricultural and Forestry Experiment Station, Mississippi State University, Mississippi State, MS 39762

Although light has been known to enhance certain toxic reactions since 1888, the principle was not exploited until after 1970 to any great extent. The greatest concentration of effort has been in the study of photodynamically active dyes, p r i marily the halogenated fluorescein series, as prospective insecticides. More recently, compounds of plant origin have been isolated, identified, and studied as phototoxins against a wide range of pests, including insects, fungi, and weeds. The main classes studied to this time are the furanocoumarins, thiophenes, acetylenes, extended quinones, and the chlorophyll a intermediates popularized as "laser herbicides." It is apparent that this area of research will expand in the coming years rather than retrench.

The e x p e n d i t u r e o f energy f r e q u e n t l y h e l p s t o enhance the proba b i l i t y o f s u c c e s s f u l l y r e a c h i n g one's g o a l s i n t h i s u n i v e r s e . F o r as long as c h e m i s t r y has e x i s t e d as a s c i e n c e , we have i n p u t energy, most f r e q u e n t l y heat energy, i n t o c h e m i c a l r e a c t i o n s t o make the m o l e c u l e s o r t o produce the e f f e c t s which we wanted. The use o f l i g h t energy has remained q u a n t i t a t i v e l y a minor component as a means o f energy i n p u t . T h i s has a l s o been the case w i t h the development o f the p e s t i c i d e i n d u s t r y . L i g h t energy has not been used to d r i v e t o x i c o l o g i c a l r e a c t i o n s o r t o p r o v i d e s p e c i f i c i t y f o r those r e a c t i o n s t o any g r e a t e x t e n t u n t i l the decade o f the 70's. S e v e r a l r e v i e w c h a p t e r s have been w r i t t e n c o v e r i n g i n d i v i d u a l a s p e c t s o f p h o t o d y n a m i c a l l y a c t i v e p e s t i c i d e s ( 1 - 8 ) . The purpose of t h i s c h a p t e r i s t o p r o v i d e a c h r o n o l o g i c a l treatment o f the development o f l i g h t as an i n t e g r a l p a r t o f the t o x i c o l o g i c a l a c t i o n o f s e v e r a l c l a s s e s o f p e s t i c i d e s ; and a l s o , t o show the development o f the v a r i o u s c l a s s e s o f l i g h t a c t i v a t e d p e s t i c i d e s r e l a t i v e t o each o t h e r . 0097-6156/87/0339-0001 $06.25/0 © 1987 American Chemical Society

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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LIGHT-ACTIVATED PESTICIDES


Early History The f i r s t documented s t u d y i n which l i g h t was understood t o cause an enhancement o f a c h e m i c a l l y induced t o x i c e f f e c t was t h a t o f M a r c a c c i (9^) i n which he r e p o r t e d t h a t a l k a l o i d s were more e f f e c t i v e a g a i n s t seeds, p l a n t s , f e r m e n t a t i o n s , and amphibian eggs i n s u n l i g h t than i n the dark. Rabb (10) s u b s e q u e n t l y r e p o r t e d t h a t s u n l i g h t caused an i n c r e a s e of s e v e r a l o r d e r s o f magnitude i n the a c r i d i n e s e n s i t i z e d m o r t a l i t y o f paramecia. Paramecia exposed t o a c r i d i n e i n the dark and paramecia exposed t o the sun i n c l e a r water were not n e a r l y as v u l n e r a b l e . By 1904, J o d l b a u e r and von Tappeiner (1_1) had demonstrated the requirement f o r oxygen and had c o i n e d the term "photodynamic a c t i o n . " Much l a t e r , S p i k e s and G l a d (12) would o p e r a t i o n a l l y d e f i n e photodynamic a c t i o n as the k i l l i n g or damaging o f an organism, c e l l , or v i r u s or the c h e m i c a l m o d i f i c a t i o n o f a m o l e c u l e i n the presence o f a s e n s i t i z i n g dye and molec u l a r oxygen. One problem i n the e a r l y development o f l i g h t a c t i v a t i o n o f m o l e c u l e s as a c o n t r i b u t o r t o a d e l e t e r i o u s e f f e c t or even the death o f a l i v i n g specimen was t h a t l i g h t was not cons i d e r e d as an e x p e r i m e n t a l parameter. Therefore, i t i s d i f f i c u l t to scan the e a r l y l i t e r a t u r e f o r examples s i m p l y because the l i g h t i n t e n s i t y was u s u a l l y u n c o n t r o l l e d and u n r e p o r t e d (13-27). The f i r s t r e p o r t e d use o f photodynamic a c t i o n a g a i n s t an i n s e c t t a r g e t was t h a t o f B a r b i e r i (28) i n which Anopheles and C u l e x mosquito l a r v a e were shown to be s u s c e p t i b l e t o s o l u t i o n s o f s e v e r a l c l a s s e s o f dyes i n d i r e c t s u n l i g h t . The most a c t i v e dyes were the h a l o g e n a t e d f l u o r e s c e i n d e r i v a t i v e s , e r y t h r o s i n and rose b e n g a l , alone and i n m i x t u r e ( I ) . There were no deaths r e p o r t e d from e i t h e r d y e - t r e a t e d , n o n - l i g h t - e x p o s e d p o p u l a t i o n s or non-dyetreated, light-exposed populations. The approach l a y dormant u n t i l 1950, when Schildmacher (29) t r e a t e d Anopheles and Aedes mosquito l a r v a e w i t h a s e r i e s o f dye s o l u t i o n s and exposed them t o s u n l i g h t . C o n d u c t i n g f i e l d t e s t s i n s m a l l ponds and a t l e a s t on bomb c r a t e r l e f t over from World War I I as w e l l as i n l a b o r a t o r y t e s t s , he r e p o r t e d t h a t rose b e n g a l was more t o x i c than e r y t h r o s i n and t h a t e o s i n and f l u o r e s c e i n were i n e f f e c t i v e . S c h i l d m a c h e r a l s o made the f i r s t attempt a t the d e f i n i t i o n o f the t o x i c o l o g i c a l t a r g e t when he r e p o r t e d t h a t the midgut e p i t h e l i a l c e l l s showed c o n s i d e r a b l e damage a f t e r l i g h t exposure. F i n a l l y , he observed t h a t photodynamic a c t i o n had no e f f e c t on the mosquito f i s h (Gambusia sp.) t h a t were p r e s e n t . I n o r d e r t o put these f i n d i n g s i n t o p e r s p e c t i v e , one s h o u l d be aware o f the s t a t e o f the a r t i n p e s t i c i d e t e c h n o l o g y a t t h i s t i m e . Ware (30) l i s t e d the f o l l o w i n g as some o f the i m p o r t a n t m i l e s t o n e s d u r i n g t h i s p e r i o d . Pyrethrum was i n t r o d u c e d i n t o Kenya (1928). M e t h y l bromide (1932), p e n t a c h l o r o p h e n o l (1936), TEPP (1938), B a c i l l u s t h u r i n g i e n s i s (1938), DDT ( 1 9 3 9 ) , h e x a c h l o r o c y c l o h e x a n e (1941), 2,4-D (1942), w a r f a r i n (1944), c h l o r d a n e ( 1 9 4 5 ) , toxaphene ( 1 9 4 7 ) , m a l a t h i o n (1950), and Maneb (1950) were e i t h e r d i s c o v e r e d or i n t r o d u c e d . At t h i s t i m e , the p r i m a r y c r i t e r i o n f o r a p e s t i c i d e was i t s t o x i c i t y , as R a c h e l Carson would not w r i t e " S i l e n t S p r i n g " f o r another decade.


1.

HEITZ

Photoactivated Compounds as Pesticides

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D u r i n g t h i s same time a l s o , b i o c h e m i s t s and p h o t o b i o l o g i s t s became i n t e r e s t e d i n the mechanism of dye s e n s i t i z e d p h o t o o x i d a t i o n and i t s e f f e c t on l i v i n g c e l l s and c e l l u l a r components. Many e x c e l l e n t reviews have been w r i t t e n on the s u b j e c t (31-37). P h o t o s e n s i t i z a t i o n has been shown to occur by one of two mechanisms: Type I and Type I I . The i n i t i a l s t e p i n the p h o t o s e n s i t i z a t i o n process i s the a b s o r p t i o n of v i s i b l e o r UV l i g h t by the s e n s i t i z e r . I n the Type I mechanism, the e x c i t e d s e n s i t i z e r conv e r t s the s u b s t r a t e t o product v i a f r e e r a d i c a l i n t e r m e d i a t e s i n c l u d i n g oxygen. In the Type I I mechanism, the e x c i t e d s e n s i t i z e r r e a c t s by c a u s i n g the f o r m a t i o n of s i n g l e t oxygen which then r e a c t s w i t h the s u b s t r a t e , t h e r e b y c o n v e r t i n g i t t o the o x i d i z e d p r o d u c t . Dye I n s e c t i c i d e s The concept l a y dormant a g a i n u n t i l 1971, when a group at West V i r g i n i a U n i v e r s i t y , Yoho, B u t l e r , and Weaver, then p u b l i s h e d the f i r s t of s e v e r a l i n v e s t i g a t i o n s of the e f f i c a c y of photodynamic a c t i o n a g a i n s t the a d u l t house f l y u s i n g p r i m a r i l y the halogenated f l u o r e s c e i n s e r i e s of dyes ( 3 8 ) . These p a p e r s , based s u b s t a n t i a l l y on the d i s s e r t a t i o n o f Yoho ( 1 9 ) , compared t o x i c o l o g i c a l d a t a w i t h the parameters o f l i g h t source and i n t e n s i t y , dye s t r u c t u r e and c o n v e n t r a t i o n i n the d i e t , source o f l i g h t , and l e n g t h of l i g h t exposure (38,40). L a t e r , Yoho et_ a l . (41) s t u d i e d a s e r i e s of 14 Food, Drug and Cosmetic dyes f o r e f f i c a c y i n photodynamic t o x i c i t y to house f l y a d u l t s . I t was a l s o r e p o r t e d i n the d i s s e r t a t i o n t h a t the midgut e p i t h e l i a l c e l l s appeared t o be damaged and t h a t the e x t e r n a l symptoms a s s o c i a t e d w i t h t o x i c i t y suggested an involvement w i t h the nervous system. I t can f a i r l y be s a i d t h a t the g r e a t m a j o r i t y of the work on p h o t o s e n s i t i z i n g dyes as i n s e c t i c i d e s can be t r a c e d back to the f i r s t paper i n t h i s s e r i e s as the watershed. A f t e r i t s p u b l i c a t i o n , t h e r e came a deluge o f i n t e r e s t i n t h i s area. Graham e^t a l . (42) r e p o r t e d t h a t w i t h the methylene b l u e sens i t i z e d p h o t o t o x i c i t y o f y e l l o w mealworms, the i n t e n s i t y of s u n l i g h t was much more than r e q u i r e d t o o b t a i n adequate e f f e c t i v e n e s s . Yoho e t a l . (40) a t t r i b u t e d the lower t o x i c i t y o f methylene blue ( I I ) i n f l u o r e s c e n t l i g h t r e l a t i v e to s u n l i g h t to the poor o v e r l a p w i t h the a b s o r p t i o n spectrum i n the former case. Broome ej: a_l. (43,44) r e p o r t e d on the t o x i c i t y o f a s e r i e s o f xanthene dyes w i t h the b l a c k imported f i r e ant where m o r t a l i t y was compared w i t h dye s t r u c t u r e , i n c u b a t i o n p e r i o d i n c o n t a c t w i t h the dye, dye c o n c e n t r a t i o n i n the feed and i n the i n s e c t , c o n t i n u i t y o f l i g h t exposure, l i g h t i n t e n s i t y , and exposure time. A l t h o u g h t h e r e was no m o r t a l i t y observed i n the imported f i r e ant a f t e r 3 days o f exposure to rose b e n g a l i n the d a r k , they d i d observe an onset o f m o r t a l i t y t h a t e v e n t u a l l y r e s u l t e d i n an L T 5 0 v a l u e of 8.4 days. T h i s may be compared w i t h an L T 5 0 v a l u e of 0.7 h r f o r a d u l t f i r e ants exposed to 3800yUW/cm2 from a C o o l White f l u o r e s c e n t l i g h t a f t e r 24 h r exposure to the r o s e bengal i n the dark (Broome et^ a l . (44). T h i s o b s e r v a t i o n l e d t o the acceptance o f the dark r e a c t i o n as a second, though a d m i t t e d l y much l e s s e f f i c i e n t , t o x i c mechanism caused by c e r t a i n photodynamic dyes i n i n s e c t s . Q u a n t i t a t i v e study of the dark r e a c t i o n w i t h a d u l t l i f e stages of the b o l l w e e v i l




B

A

B

H Br I Br I

H H H

a

CI

DYE Fluorescein Eosin Erythrosin B Phloxin B Rose Bengal

Structure I

Methylene Blue

Structure I I



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( 4 5 ) , the face f l y ( 4 6 ) , and the house f l y (47) showed the w i d e s p r e a d o c c u r r a n c e of t h i s t o x i c mechanism. In f a c t , D a v i d and H e i t z (48) r e p o r t e d on an imported f i r e ant f i e l d c o n t r o l scheme based on a p h l o x i n B-impregnated b a i t where the c o n t r o l r e p o r t e d was almost c e r t a i n l y due to the dark mechanism w o r k i n g deep w i t h i n the n e s t . At about t h i s same time, mechanism s t u d i e s were a p p e a r i n g . The a c e t y l c h o l i n e s t e r a s e from the b l a c k imported f i r e ant (49) and the b o l l w e e v i l (J>0) was s u s c e p t i b l e t o d y e - s e n s i t i z e d p h o t o o x i d a t i o n i n v i t r o but l e v e l s were not depressed i n i n s e c t s k i l l e d by photodynamic a c t i o n . Weaver e£ a l . (_51) r e p o r t e d t h a t i n the c o c k r o a c h , photodynamic a c t i o n caused a s i g n i f i c a n t decrease i n the hemolymph volume and a l a r g e i n c r e a s e i n the c r o p volume. L a t e r , Weaver ej: a l . (52) showed t h a t e r y t h r o s i n B - s e n s i t i z e d photodynamic a c t i o n caused a r e d u c t i o n of hemocytes r e l a t i v e to c o n t r o l s . At the h i g h e s t i n j e c t e d l e v e l s i n the d a r k , t h e r e was a l s o observed a r e d u c t i o n i n hemocytes, which i s p r o b a b l y due to the dark t o x i c mechanism. I n the absence of l i g h t , Broome et_ a l . (53) reported t h a t i n b o l l w e e v i l s fed r o s e bengal d u r i n g l a r v a l development, t h e r e were decreases i n the wet w e i g h t , dry w e i g h t , p r o t e i n l e v e l s , and l i p i d l e v e l s of the a d u l t i n s e c t . L a t e r , Callaham et^ a l . (54) showed t h a t the lower l e v e l s were due t o a l a c k of growth a f t e r a d u l t emergence i n the t r e a t e d i n s e c t s . T h i s was i n t e r p r e t e d as an energy d r a i n caused by the presence of the dye i n the a d u l t t i s s u e . In 1978, Fondren et^ al^. (47) compared the t o x i c i t i e s o f 6 xanthene dyes to the house f l y i n terms of both d i e t a r y and t i s s u e l e v e l s of the dyes i n q u e s t i o n . I n d i c a t i o n s of f e e d i n g i n h i b i t i o n were observed a t h i g h dye c o n c e n t r a t i o n s i n the food. Although s p e c i e s d i f f e r e n c e s were observed when the house f l y d a t a was compared w i t h s i m i l a r b o l l w e e v i l d a t a , i t was r e p o r t e d t h a t , i n g e n e r a l , the e f f e c t i v e n e s s of the dyes was most dependent on the phosphorescence quantum y i e l d than any o t h e r p h y s i c o - c h e m i c a l parameter. S i m i l a r i n t e r p r e t a t i o n s were made i n a l a t e r s t u d y o f the face f l y ( 4 6 ) . I n a s t u d y of l i g h t i n t e n s i t y as a c r i t i c a l parameter i n the photodynamic t o x i c i t y of rose b e n g a l to the a d u l t house f l y , Fondren and H e i t z (55) showed t h a t the accumulated number of photons needed to k i l l 50% o f a p o p u l a t i o n decreased as the i n t e n s i t y increased. T h i s would i n f e r t h a t t h e r e i s a r e g e n e r a t i v e c a p a c i t y w i t h i n the i n s e c t t h a t i s more e f f i c i e n t l y overcome by photodynamic a c t i o n as the l i g h t i n t e n s i t y i n c r e a s e s . L i g h t source was a l s o s t u d i e d as an e x p e r i m e n t a l parameter (56) where i t was shown t h a t s u n l i g h t was more e f f i c i e n t than f l u o r e s c e n t l i g h t due to the l a r g e r number of photons s t r i k i n g the t a r g e t ; but i t was a l s o shown t h a t f l u o r e s c e n t l i g h t was more e f f i c i e n t than s u n l i g h t due to the b e t t e r o v e r l a p o f the lamp output w i t h the a b s o r p t i o n spectrum of the xanthene dyes. L a v i a l l e and Dumortier (57) r e p o r t e d t h a t methylene b l u e - f e d l a r v a e of the cabbage b u t t e r f l y were s u s c e p t i b l e t o photodynamic a c t i o n a f t e r exposure to e i t h e r f l u o r e s c e n t l i g h t or s u n l i g h t . M o r t a l i t y was shown to be dependent on dye c o n c e n t r a t i o n , l i g h t i n t e n s i t y , d u r a t i o n , and w a v e l e n g t h . I n l a b o r a t o r y t o x i c i t y t e s t s u s i n g s e v e r a l xanthene dyes a g a i n s t the b l a c k cutworm, Clement et a l . (58) found t h a t rose b e n g a l was


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the most e f f e c t i v e and t h a t t o x i c i t y was d i r e c t l y dependent on the light intensityI n t h i s c a s e , the l a r v a e a v o i d s the l i g h t and t h a t makes t h i s p a r t i c u l a r a p p l i c a t i o n u n d e s i r e a b l e . C r e i g h t o n et a l . (59) r e p o r t e d on the t o x i c i t y of rose bengal t o the cabbage l o o p e r , the c o r n earworm, and the p i c k l e w o r m . Photodynamic a c t i o n was r e l a t i v e l y i n e f f e c t i v e under these c o n d i t i o n s , but the dark t o x i c i t y was observed. I n 1979, P i m p r i k a r ^ t a l . (60) began r e p o r t i n g on an extended s e r i e s o f t e s t s w i t h mosquito l a r v a e . Under f l u o r e s c e n t l i g h t and at r o s e bengal treatment l e v e l s of 1 t o 20 ppm, C u l e x l a r v a e were more s u s c e p t i b l e than Aedes l a r v a e and e a r l y i n s t a r s were more s u s c e p t i b l e than l a t e r i n s t a r s . P h y s i o l o g i c a l and m o r p h o l o g i c a l a b n o r m a l i t i e s were observed i n the pupal and a d u l t stage a f t e r l a r v a l stage treatment which suggested improper c h i t i n f o r m a t i o n i n the i n s e c t . T h i s sometimes r e s u l t e d i n i n c o m p l e t e e x t r i c a t i o n o f the pupal stage from the l a r v a l c u t i c l e and of the a d u l t stage from the pupal c u t i c l e . Where t h i s was o b s e r v e d , m o r t a l i t y r e s u l t e d . They a l s o r e p o r t e d the observance of l a r v a l - p u p a l i n t e r m e d i a t e s s i m i l a r t o those observed a f t e r treatment w i t h i n s e c t growth r e g u lators • P i m p r i k a r et^ al. (61) attempted t o c o n t r o l house f l i e s i n a commercial caged l a y e r house u s i n g weekly a p p l i c a t i o n s of aqueous s o l u t i o n s of e r y t h r o s i n B d i r e c t l y on the manure. I n a d u p l i c a t e d 5 week treatment p e r i o d , they r e p o r t e d decreases o f a d u l t and l a r v a l house f l i e s up t o 90% w i t h r e s p e c t t o p r e t r e a t m e n t l e v e l s w i t h no change i n the b e n e f i c i a l s o l d i e r f l y l a r v a l p o p u l a t i o n . The dye was r e p o r t e d t o be somewhat r a p i d l y degraded i n the manure i l l u minated by i n d i r e c t s u n l i g h t such t h a t o n l y about 20% was e x t r a c t a b l e 1 week a f t e r s p r a y i n g . As a r e s u l t o f these t e s t s , P i m p r i k a r et a l . (62) s t u d i e d the e f f e c t s of s e v e r a l f l u o r e s c e i n d e r i v a t i v e s on each developmental stage o f the house f l y . Treated adults e x h i b i t e d lowered f e c u n d i t y , the eggs e x h i b i t e d a reduced v i a b i l i t y , and m o r t a l i t y was observed i n each l i f e stage o f the house f l y . C a r p e n t e r and H e i t z (63) showed t h a t when l a r v a l mosquitoes were exposed t o r o s e bengal and v i s i b l e l i g h t , s i g n i f i c a n t acute m o r t a l i t y was observed. F u r t h e r , i f the t r e a t e d mosquitoes were i l l u m i n a t e d w i t h v i s i b l e l i g h t and then put i n t o d a r k n e s s , a l a t e n t m o r t a l i t y was observed. The l i g h t treatment was n e c e s s a r y t o o b t a i n the l a t e n t m o r t a l i t y , as the c o n t r o l s exposed t o the same dye c o n c e n t r a t i o n s i n the dark e x h i b i t e d no l a t e n t m o r t a l i t y . When the l a t e n t m o r t a l i t y was added t o the acute m o r t a l i t y , i t was observed t h a t the t o t a l t o x i c i t y o f the rose bengal was i n c r e a s e d by 1 0 - f o l d over the dark t o x i c i t y . L a t e r , C a r p e n t e r and H e i t z (64) s t u d i e d the r e l a t i o n s h i p s between the slow, l i g h t - i n d e p e n d e n t mechanism, the r a p i d , l i g h t - d e p e n d e n t mechanism, and the s l o w , l i g h t - i n i t i a t e d , l a t e n t mechanism d u r i n g the treatment of C u l e x l a r v a e w i t h e r y t h r o s i n B. Q u a n t i t a t i v e a n a l y s i s was hampered by the p h o t o d e g r a d a t i o n o f the e r y t h r o s i n B d u r i n g the time course o f the study which made the e x p r e s s i o n of t o x i c i t y r e l a t i v e t o dye concentration impossible.



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F a i r b r o t h e r et a l . (j>5) made a v e r y complete study o f the t o x i c o l o g i c a l e f f e c t s o f e r y t h r o s i n B and rose bengal on the face f l y When l a r v a e developed on manure i n t o which e i t h e r dye was i n c o r p o r a t e d , e i t h e r by hand or by passage o f the dye through c a t t l e , m o r t a l i t y was observed at each l i f e s t a g e . Some of the f l i e s d i e d at v a r i o u s stages o f emergence as i f the e f f o r t a s s o c i a t e d w i t h emergence was too s t r e s s f u l . S e v e r a l of the a d u l t s were unable to complete the e x t r i c a t i o n from the puparium and were s t u c k t o the c h i t i n i n n e r l i n i n g of the puparium. Others had deformed wings. A d u l t s , h e l d from emergence and i l l u m i n a t e d w i t h v i s i b l e l i g h t , were observed to have a much h i g h e r m o r t a l i t y than c o n t r o l s , thus s u g g e s t i n g t h a t dye s e q u e s t e r e d i n the i n s e c t body d u r i n g d e v e l o p ment from l a r v a e to a d u l t was r e s p o n s i b l e f o r the t o x i c i t y . This i s the f i r s t r e p o r t of photodynamic a c t i o n o c c u r r i n g i n a l i f e stage d i f f e r e n t from the l i f e stage which i n g e s t e d the dye. C a r p e n t e r ejt al. (66) r e p o r t e d t h a t the presence of f l u o r e s c e i n enhanced the t o x i c i t y of r o s e bengal i n photodynamic r e a c t i o n s i n Aedes l a r v a e . T h i s s y n e r g i s m was i n i t i a l l y e x p l a i n e d by the u t i l i z a t i o n of photons absorbed by the f l u o r e s c e i n m o l e c u l e t h a t were not of the proper wavelength f o r a b s o r p t i o n by the r o s e b e n g a l m o l e c u l e . A U n i t e d S t a t e s p a t e n t was i s s u e d c o v e r i n g the s y n e r g i s m of a n o n t o x i c dye w i t h a demonstrated t o x i c dye i n b o t h house f l y and mosquito systems (6i7). L a t e r , i n t e s t s i n v o l v i n g 8 xanthene dyes, i t was not p o s s b i l e to c o n f i r m the mechanism of a c t i o n as t h a t r e f e r r e d t o above ( 6 8 ) . F u r t h e r , the s y n e r g i s m c o u l d not be c o r r e l a t e d w i t h the number of h a l o g e n s , p e r c e n t h a l o g e n a t i o n , molec u l a r w e i g h t , p a r t i t i o n c o e f f i c i e n t , f l u o r e s c e n c e quantum y i e l d o f the s y n e r g i s t dye, or the o v e r l a p i n t e r v a l f o r the s y n e r g i s t dye w i t h e r y t h r o s i n B. The mechanism o f a c t i o n of the s y n e r g i s m observed w i t h the xanthene dyes i s s t i l l u n e x p l a i n e d . S a k u r a i and H e i t z (69) s t u d i e d the i n h i b i t i o n of growth and the photodynamic a c t i o n caused by r o s e b e n g a l and e r y t h r o s i n B i n the house f l y . L a r v a e r e a r e d i n the dark on agar c o n t a i n i n g e i t h e r dye e x h i b i t e d a c o n c e n t r a t i o n dependent decrease i n p u p a t i o n r a t e and i n a d u l t emergence. House f l i e s which had consumed a n o n l e t h a l amount of dye i n the l a r v a l stage e x h i b i t e d a c o n s i d e r a b l e l i g h t dependent t o x i c i t y i n the a d u l t s t a g e . I t was a l s o observed t h a t pupae i n j e c t e d w i t h rose b e n g a l developed i n t o a d u l t s which were more s u s c e p t i b l e to photodynamic a c t i o n than a d u l t s i n j e c t e d w i t h the same dye. F u r t h e r , the s u s c e p t i b i l i t y of the i n j e c t e d a d u l t s was comparable to a d u l t s fed the dye, thus s u g g e s t i n g t h a t the a l i mentary c a n a l may not be the o n l y s i t e o f a c t i o n as suggested p r e v i o u s l y (29,39,51). I n 1983, R e s p i c i o and H e i t z (70) began a study of the d e v e l o p ment of r e s i s t a n c e to e r y t h r o s i n B i n the house f l y . A l a b o r a t o r y s t r a i n developed o n l y 6 - f o l d r e s i s t a n c e a f t e r 40 g e n e r a t i o n s of c h a l l e n g e by e r y t h r o s i n B. T h i s low l e v e l o f r e s i s t a n c e was due to the i n b r e d q u a l i t y o f the l a b o r a t o r y s t r a i n . A new, w i l d s t r a i n developed a 4 8 - f o l d r e s i s t a n c e a f t e r 32 g e n e r a t i o n s of exposure to i n c r e a s i n g l e v e l s of e r y t h r o s i n B i n the d i e t . Upon removal of the s e l e c t i o n p r e s s u r e f o r 20 g e n e r a t i o n s , the r e s i s t a n c e remained constant. R e c i p r o c a l c r o s s e s showed t h a t the r e s i s t a n c e i s i n h e r i t e d as a codominant c h a r a c t e r and t h a t sex l i n k a g e i s not


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i n v o l v e d . L a t e r , the c r o s s - r e s i s t a n c e of e r y t h r o s i n B - r e s i s t a n t house f l i e s was s t u d i e d a g a i n s t s t r a i n s r e s i s t a n t to propoxur, DDT, p e r m e t h r i n , and d i c h l o r v o s (71). No c r o s s - r e s i s t a n c e f o r a d i f f e r e n t p e s t i c i d e was observed f o r any of the 5 s t r a i n s , w i t h one exception. The e r y t h r o s i n B - r e s i s t a n t s t r a i n was c r o s s - r e s i s t a n t to p h l o x i n B and r o s e b e n g a l , but t h i s i s t o be expected s i n c e they f u n c t i o n by the same mechanism. R e c e n t l y , c r o s s - r e s i s t a n c e has been shown when the e r y t h r o s i n B - r e s i s t a n t s t r a i n was c h a l l e n g e d by a l p h a - t e r t h i e n y l mediated photodynamic a c t i o n ( P i m p r i k a r , G.D. and H e i t z , J.R., u n p u b l i s h e d r e s u l t s ) . The r e l a t i v e t o x i c i t i e s of 6 xanthene dyes t o C u l e x and Aedes mosquito l a r v a e was r e p o r t e d by P i m p r i k a r e t a l . ( 7 2 ) . Rose b e n g a l was the most t o x i c f o l l o w e d by p h l o x i n B and e r y t h r o s i n B. At the same t i m e , i t was shown t h a t these same dyes e x h i b i t e d a low t o x i c i t y t o the mosquito f i s h , t h e r e b y c o n f i r m i n g the o b s e r v a t i o n o f Schildmacher ( 2 9 ) , and d i d not a f f e c t the p r e d a t o r y e f f i c i e n c y o f the f i s h . The l a c k of an e f f e c t on the p r e d a t o r y mosquito f i s h would a l l o w the dyes t o be c o n s i d e r e d i n an i n t e g r a t e d p e s t management scheme. I n 1984, P i m p r i k a r and H e i t z (73) observed an u n u s u a l l y h i g h i n s e c t i c i d a l a c t i v i t y i n Aedes mosquito l a r v a e which had been i l l u minated a f t e r exposure to the i n s o l u b l e f r e e a c i d forms o f the xanthene dyes. I n a l l p r e v i o u s s t u d i e s , the l a r v a e had been t r e a t e d w i t h the water s o l u b l e s a l t forms o f the dyes and the l a r v a e consumed the dye as they i n g e s t e d the w a t e r . W i t h the i n s o l u b l e dyes, they were a b l e t o f i l t e r feed on dye p a r t i c l e s and t h e r e b y r e c e i v e a h i g h e r l e v e l of dye. T o x i c i t y r a t i o s ranged up to 2 o r d e r s of magnitude between the s o l u b l e and i n s o l u b l e forms of the same dye. In a l a t e r s t u d y , C a r p e n t e r et a l . (74) showed t h a t the i n s o l u b l e forms of the xanthene dyes were 1 0 - f o l d more e f f e c t i v e a g a i n s t C u l e x mosquito l a r v a e than the s o l u b l e forms. F u r t h e r , they r e p o r t e d t h a t when the i n s o l u b l e forms o f the dyes were d i s p e r s e d w i t h a s u r f a c t a n t , such as sodium l a u r y l s u l f a t e , the dyes were 50- t o 6 0 - f o l d more e f f e c t i v e than the s o l u b l e forms. R e s p i c i o et a l _ . (75^) s t u d i e d the t o x i c i t y t o C u l e x mosquito l a r v a e of c o p r e c i p i t a t e d f r e e a c i d , n o n d i s p e r s i b l e and d i s p e r s i b l e f o r m u l a t i o n s of f l u o r e s c e i n and e r y t h r o s i n B. The 1:1 c o m b i n a t i o n o f f l u o r e s c e i n : e r y t h r o s i n B, d i s p e r s e d w i t h sodium d o d e c y l s u l f a t e , was the most t o x i c f o r m u l a t i o n and a l s o showed s y n e r g i s t i c a c t i o n . I n a more d e t a i l e d study of the s y n e r g i s t i c e f f e c t , they showed t h a t the 1:1 m i x t u r e of f l u o r e s c e i n : r o s e b e n g a l was more t o x i c than the 3:1 m i x t u r e , but the 3:1 m i x t u r e e x h i b i t e d more s y n e r g i s m ( R e s p i c i o , N.C., C a r p e n t e r , T.L., and H e i t z , J.R. J . M i s s . Acad. S c i , i n p r e s s ) . C a r p e n t e r e t a l . (76) e v a l u a t e d a s e r i e s o f 8 d i s p e r s a n t s f o r use w i t h the i n s o l u b l e forms o f the dyes and none were t o x i c a l o n e . E r y t h r o s i n B, d i s p e r s e d w i t h sodium d o d e c y l s u l f a t e , was the most t o x i c a g a i n s t C u l e x mosquito l a r v a e . In s m a l l - s c a l e f i e l d t e s t s , t h i s f o r m u l a t i o n caused s i g n i f i c a n t r e d u c t i o n s i n l a r v a l and emergent a d u l t p o p u l a t i o n s o f C u l e x mosquitoes a t c o n c e n t r a t i o n s r a n g i n g from 0.25 t o 8.0 ppm. Not a l l of the work w i t h the f l u o r e s c e i n dyes concerned i n s e c t s . In 1985, Knox and Dodge (7J7) r e p o r t e d on the photodynamic



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a c t i o n of e o s i n on pea l e a f t i s s u e . C h l o r o p l a s t s were shown t o be p a r t i c u l a r l y s e n s i t i v e to v i s i b l e l i g h t a f t e r e o s i n t r e a t m e n t . The t r e a t e d t i s s u e e x h i b i t e d lowered p h o t o s y n t h e t i c oxygen e v o l u t i o n , lowered p h o t o s y n t h e t i c e l e c t r o n t r a n s p o r t c a p a b i l i t y , lowered l e v e l s of r i b u l o s e - b i s p h o s p h a t e c a r b o x y l a s e and NADPH-dependent g l y c e r a l d e h y d e - 3 - p h o s p h a t e dehydrogenase, and pigment l o s s . The i n i t i a l l o s s of p h o t o s y n t h e t i c a c t i v i t y was a s s o c i a t e d w i t h damage t o the t h y l a k o i d membranes. In an accompanying paper, Knox and Dodge (78) f u r t h e r c h a r a c t e r i z e d the s i t e of the photodynamic a c t i o n i n pea l e a f t i s s u e as photosystem I I . Robins and Beatson (_79) attempted t o p r o t e c t house f l y l a r v a e against e r y t h r o s i n B s e n s i t i z e d p h o t o t o x i c i t y . Beta-carotene prot e c t e d , but b u t y l a t e d h y d r o x y t o l u e n e , a s c o r b a t e , and d i a z a b i c y c l o o c t a n e a c t u a l l y enhanced the t o x i c e f f e c t . Hawkins et_ a l . (80) showed t h a t e r y t h r o s i n B and v i s i b l e l i g h t (from e i t h e r f l u o r e s c e n t sources or s u n l i g h t ) were t o x i c to the i n f e c t i o u s 3rd stage l a r v a e o f g a s t r o i n t e s t i n a l nematodes of n a t u r a l l y i n f e c t e d c a t t l e . T o x i c i t y was s i g n i f i c a n t a f t e r o n l y 2 c o n s e c u t i v e d a i l y o r a l t r e a t m e n t s of the c a t t l e . L a t e r , they r e p o r t e d t h a t the photodynamic a c t i o n was i n e f f e c t i v e a g a i n s t the a d u l t stage v i a b i l i t y or f e c u n d i t y (Hawkins, J.A.; J o h n s o n - D e l i v o r i a s , M.H.; H e i t z , J.R. Veterin. Parasitol., in p r e s s ) . There was a c o n s i s t e n t e f f e c t on the 3rd stage l a r v a e which was dependent upon dosage, time of l i g h t exposure and, t o a l e s s e r e x t e n t , the l e n g t h o f time the l a r v a e were l e f t i n the presence of the dye. P h o t o a c t i v e P l a n t Components I n the study of the p h o t o a c t i v e dyes, the r e s e a r c h was focused p r i m a r i l y on a deep u n d e r s t a n d i n g of the mechanisms of a c t i o n of o n l y a s m a l l number of dyes from p r e d o m i n a t e l y one c l a s s o f compounds. I n the study o f p h o t o a c t i v e p l a n t components, t h e r e has been a s h i f t of emphasis. Much of the r e s e a r c h has been aimed a t i s o l a t i o n and i d e n t i f i c a t i o n of n o v e l p l a n t components, d e l i n e a t i o n of the g e n e r a l mechanisms of a c t i o n and the type of s e n s i t i v e organism. As such, t h e r e are fewer papers on any g i v e n compound, but many more compounds s t u d i e d . Some o f the major c l a s s e s o f p l a n t d e r i v e d compounds w i l l be examined h e r e . R e c e n t l y , an e n t i r e i s s u e of the J o u r n a l of Chemical E c o l o g y was devoted t o the i n v i t e d papers p r e sented a t a symposium on i n t e r a c t i o n s between i n s e c t s and photoa c t i v e p l a n t s p r e s e n t e d a t the 1984 n a t i o n a l meeting o f the E n t o m o l o g i c a l S o c i e t y of America ( 8 1 ) . S e v e r a l r e l a t e d papers were a l s o i n c l u d e d which were not p a r t o f the symposium. Furanocoumarins Furanocoumarin8 have been i m p l i c a t e d i n c e r t a i n p h o t o t o x i c r e s p o n ses i n g r a z i n g c a t t l e ( 8 2 ) . I n 1978, Berenbaum (83) r e p o r t e d t h a t when the l i n e a r furanocoumarin, x a n t h o t o x i n ( I I I ) , was a d m i n i s t e r e d to the l a r v a e of the s o u t h e r n armyworm, a low l e v e l of t o x i c i t y was observed t h a t was g r e a t l y enhanced when UV l i g h t was shown upon the l a r v a e . She a l s o observed a l o n g e r time r e q u i r e d f o r p u p a t i o n t o



10


o c c u r i n those l a r v a e t h a t d i d n o t d i e - The b i o l o g i c a l a c t i v i t y o f the furanocoumarins a r e due t o the i n t e r c a l c a t i o n o f the m o l e c u l e i n t o t h e double s t r a n d e d DNA where, upon a c t i v a t i o n by UV l i g h t , c o v a l e n t bonds a r e formed w i t h p y r i m i d i n e bases ( 8 4 ) . Song and T a p l e y (85) demonstrated t h a t the mechanism o f a c t i o n was Type I i n which oxygen r a d i c a l s a r e i n v o l v e d . L a t e r , Berenbaum and Feeny (86) r e p o r t e d t h a t the a n g u l a r f u r a n o c o u m a r i n , a n g e l i c i n ( I V ) , reduced the growth r a t e and the f e c u n d i t y of the l a r v a e o f the b l a c k s w a l l o w t a i l b u t t e r f l y , whereas x a n t h o t o x i n was n o t a p p r e c i a b l y t o x i c t o t h i s i n s e c t . I t was i n t e r p r e t e d t h a t t h e a n g u l a r forms o f the furanocoumarin were l a t e r e v o l u t i o n a r y developments which h e l p e d t o p r o t e c t t h e p l a n t from i n s e c t h e r b i v o r y . R e c e n t l y , I v i e et^ a l . (87) i n an i n i t i a l r e p o r t on the m e t a b o l i s m o f furanocoumarins by b l a c k s w a l l o w t a i l b u t t e r f l y l a r v a e , showed t h a t t h i s i n s e c t d e t o x i f i e s t h i s c l a s s o f compounds by m e t a b o l i s m i n t h e midgut t i s s u e p r i o r t o a b s o r p t i o n . I n t h i s manner, a p p r e c i a b l e l e v e l s o f u n m e t a b o l i z e d furanocoumarin do not e n t e r t h e body c i r c u l a t i o n . L a t e r , i t was r e p o r t e d t h a t the i n c r e a s e d p h o t o t o x i c i t y o f t h e a n g u l a r furanocoumarins r e l a t i v e t o the l i n e a r furanocoumarins was due t o a s l o w e r r a t e o f h y d r o l y s i s of the f u r a n r i n g o f the a n g u l a r d e r i v a t i v e s (88,89). Ashwood-Smith et^ al. (90) r e p o r t e d t h a t the b l a c k s w a l l o w t a i l l a r v a e were a b l e t o degrade x a n t h o t o x i n i n t o b i o l o g i c a l l y i n a c t i v e compounds. The enzyme r e a c t i o n r e q u i r e d an e l e c t r o n g e n e r a t i n g and a c c e p t i n g system s i m i l a r t o t h e mixed f u n c t i o n o x i d a s e s o f mamm a l i a n microsomes. I r i v i t r o s t u d i e s o f t h e r e l a t i v e m e t a b o l i c r a t e s o f h y d r o l y s i s o f x a n t h o t o x i n by homogenates o f l a s t stage l a r v a e o f the b l a c k s w a l l o w t a i l b u t t e r f l y and the f a l l armyworm showed t h a t the former i n s e c t h y d r o l y z e d the x a n t h o t o x i n 6 times f a s t e r than the l a t t e r i n s e c t ( 9 1 ) . Alpha-Terthienyl

and P o l y a c e t y l e n e s

A l p h a - t e r t h i e n y l (V) was shown t o be n e m a t i c i d a l by Uhlenbroek and B i j l o o (92). Gommers (93^) r e p o r t e d t h a t i r r a d i a t i o n w i t h near UV l i g h t s t r o n g l y enhanced t h e n e m a t i c i d a l a c t i v i t y o f a l p h a t e r t h i e n y l . L a t e r , Gommers and G e e r l i g s (94) showed t h a t endoparas i t i c p l a n t nematodes which had been exposed t o a l p h a - t e r t h i e n y l i n the r o o t s o f A f r i c a n m a r i g o l d s f o r 10 days were r a p i d l y k i l l e d upon exposure t o near UV l i g h t . Bakker e t a l . (95) demonstrated t h a t , upon i r r a d i a t i o n , a l p h a - t e r t h i e n y l g e n e r a t e s a r e a c t i v e oxygen spec i e s , p r o b a b l y s i n g l e t oxygen, upon which the n e m a t i c i d a l a c t i v i t y depends. Gommers et^ a l . (96) r e p o r t e d t h a t t h e i r r a d i a t i o n o f a l p h a - t e r t h i e n y l r e q u i r e d a e r o b i c c o n d i t i o n s f o r the r a p i d k i l l i n g of nematodes. I n v i t r o s t u d i e s o f enzyme i n h i b i t i o n and p r o t e c t i o n by a s e r i e s o f s i n g l e t oxygen quenchers f u r t h e r supported t h e h y p o t h e s i s t h a t the a c t i v e oxygen formed i n t h e r e a c t i o n was s i n g l e t oxygen. A l p h a - t e r t h i e n y l and p h e n y l h e p t a t r i y n e (VI) were shown t o be powerf u l t o x i c p h o t o s e n s i t i z e r s a g a i n s t f i r s t and f o u r t h i n s t a r Aedes mosquito and b l a c k f l y l a r v a e i n b o t h s u n l i g h t and UV l i g h t ( 9 7 , 9 8 ) . The mode o f a c t i o n o f a l p h a - t e r t h i e n y l was shown t o be photodynamic i n nature but that of phenylheptatriyne-type compounds was n o t as


1.

HEITZ

11


OCH

3

Xanthotoxin


Structure I I I

Angelicin

S t r u c t u r e IV

a -Terthienyl

Structure V

^Q^"C=C-C»C-C=C-CH

3

I-Phenyl-1,3,5-heptatriyne

S t r u c t u r e VI



12


c l e a r (99) . McLachlan e t a_l. (100) i n v e s t i g a t e d s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s f o r a s e r i e s of p o l y a c e t y l e n e and thiophene d e r i v a t i v e s a g a i n s t a b a c t e r i u m and a y e a s t w i t h the thiophenes b e i n g g e n e r a l l y more t o x i c than the a c e t y l e n e s . A c t i v i t y was d i r e c t l y dependent upon the number of thiophene r i n g s and a c e t y l e n e bonds. There was a p o s i t i v e c o r r e l a t i o n between p h o t o t o x i c i t y and the o c t a n o l - w a t e r p a r t i t i o n c o e f f i c i e n t ; but t h e r e was l i t t l e c o r r e l a t i o n w i t h photon a b s o r p t i o n . The dose response i n r e l a t i o n t o the l i g h t source was s t u d i e d by Arnason e t a l . ( 1 0 1 ) . A l t h o u g h a l p h a - t e r t h i e n y l e x h i b i t e d low t o x i c i t y i n the absence o f l i g h t , the enhanced t o x i c i t y t o Aedes mosquito l a r v a e upon i r r a d i a t i o n by near UV l i g h t l e d t o i t s i n v e s t i g a t i o n as a commercial l a r v i c i d e i n f i e l d t r i a l s u s i n g simul a t e d s m a l l ponds. A l p h a - t e r t h i e n y l was even more t o x i c t o the mosquito l a r v a e i n s u n l i g h t . An a c t i o n spectrum showed t h a t t h e r e was good agreement between l i g h t a b s o r p t i o n and t o x i c o l o g i c a l action. I n 1983, Kagan and Chan (102) showed t h a t b o t h phenylhept a t r i y n e and a l p h a - t e r t h i e n y l d i s p l a y e d o v i c i d a l a c t i v i t y a g a i n s t the eggs o f the f r u i t f l y i n the d a r k . They r e p o r t e d t h a t i r r a d i a t i o n by l o n g wavelength UV l i g h t enhanced the t o x i c i t y by 37and 4 3 3 3 - f o l d , r e s p e c t i v e l y . U s i n g the s i n g l e t oxygen dependent c o n v e r s i o n of adamantylidene adamantane to adamantanone, Kagan et_ a l . (103) were a b l e t o compare the r e l a t i v e s i n g l e t oxygen g e n e r a t i n g c a p a b i l i t y o f a s e r i e s o f thiophene d e r i v a t i v e s . The p o l y a c e t y l e n i c compound, c i s - d e h y d r o m a t r i c a r i a e s t e r was shown t o be o v i c i d a l t o f r e s h l y l a i d eggs of the f r u i t f l y . Upon i r r a d i a t i o n w i t h u l t r a v i o l e t l i g h t the o v i c i d a l a c t i v i t y was enhanced (104). L a t e r , Downum ej: a l . (105) r e p o r t e d t h a t the tobacco hornworm, when g i v e n a s i n g l e i n g e s t e d dose o f a l p h a - t e r t h i e n y l f o l l o w e d by exposure t o UV l i g h t , e x h i b i t e d delayed and abnormal pupal f o r m a t i o n w i t h no subsequent a d u l t emergence. T o p i c a l a p p l i c a t i o n o f a l p h a - t e r t h i e n y l f o l l o w e d by i r r a d i a t i o n w i t h near UV l i g h t a f f e c t e d b o t h the s c l e r o t i z a t i o n and m e l a n i z a t i o n of the pupal case i n l a t e r development. Kagan et^ a l . (106) demonstrated the f i r s t example of the i n a c t i v a t i o n o f a c e t y l c h o l i n e s t e r a s e in v i v o by a p h o t o a c t i v e p e s t i c i d e when they showed t h a t a l p h a - t e r t h i e n y l , as w e l l as 3 i s o m e r s , caused the i n h i b i t i o n o f t h i s enzyme i n Aedes mosquito l a r v a e upon UV l i g h t i r r a d i a t i o n . L a t e r , Reyftmann e_t a l . (107) showed t h a t a l p h a - t e r t h i e n y l e x h i b i t e d a very l o n g - l i v e d e x c i t e d t r i p l e t s t a t e which a l l o w e d i t t o r e a c t v e r y f a v o r a b l y w i t h oxygen, thereby p r o d u c i n g s i n g l e t oxygen. S i n c e i t does not r e a c t w e l l w i t h hydrogen or e l e c t r o n donors, i t appears t h a t a l p h a - t e r t h i e n y l f u n c t i o n s p r i m a r i l y as a Type I I photodynamic agent. Kagan ej: a l . (108) s t u d i e d the p h o t o t o x i c e f f e c t s o f a l p h a t e r t h i e n y l on f a t h e a d minnows and i t was found t o be at l e a s t t w i c e as potent as rotenone and n e a r l y as potent as e n d r i n . I n 1985, a Canadian p a t e n t was awarded to Towers et_ a l . (109) c o v e r i n g the c o n t r o l of p e s t s ( a l g a e , f u n g i , nematodes, or h e r b i v o r o u s i n v e r t e b r a t e s ) by p o l y a c e t y l e n e s • A c t i v a t i o n of the p o l y a c e t y l e n e by the UV component of s u n l i g h t enhanced the t o x i c e f f e c t s observed i n the absence o f l i g h t .


1.

HEITZ



H y p e r i c i n and

13

Cercosporin

I t has been known f o r many y e a r s , t h a t when g r a z i n g animals feed on c e r t a i n members of the p l a n t genus Hypericum, they become s e n s i t i v e t o s u n l i g h t . T h i s s e n s i t i v i t y i s accompanied by i n t e n s e s k i n i r r i t a t i o n and i n f l a m m a t i o n which may become f a t a l . H o r s l e y (110) showed t h a t t h i s c o n d i t i o n was caused by h y p e r i c i n ( V I I ) , a h i g h l y condensed quinone (111,112). Yamazaki e_t a_l. (113) noted the s i m i l a r i t i e s between the s t r u c t u r e s of h y p e r i c i n ( V I I ) and c e r c o s p o r i n ( V I I I ) . When they exposed c e r c o s p o r i n - t r e a t e d mice and b a c t e r i a t o l i g h t , m o r t a l i t y was observed. C e r c o s p o r i n a l s o was shown t o damage p l a n t t i s s u e under i l l u m i n a t i o n by i n c a n d e s c e n t l i g h t ( 1 1 4 ) . Daub (115) r e p o r t e d t h a t the k i n e t i c s of the k i l l i n g of tobacco p l a n t c e l l s was a f u n c t i o n o f c e r c o s p o r i n c o n c e n t r a t i o n , l i g h t i n t e n s i t y , l i g h t w a v e l e n g t h , and s i n g l e t oxygen quenchers. S i n c e the t o x i c response was i n h i b i t e d by Dabco and b i x i n , known quenchers of s i n g l e t oxygen, i t was proposed t h a t c e r c o s p o r i n produced s i n g l e t oxygen and f u n c t i o n e d as a photodynamic agent. L a t e r , Daub (116) showed t h a t c e r c o s p o r i n - c a u s e d e l e c t r o l y t e leakage from tobacco l e a f d i s c s was p r o b a b l y due t o l i p i d h y d r o p e r o x i d e f o r m a t i o n from membrane l i p i d s . Cercosporin was shown to o x i d i z e s o l u t i o n s of methyl l i n o l e n a t e , w h i l e a l p h a t o c o p h e r o l had an i n h i b i t o r y e f f e c t on the c e r c o s p o r i n - m e d i a t e d l i p i d p e r o x i d a t i o n . Daub and B r i g g s (117) then showed t h a t the u n s a t u r a t e d a c y l c h a i n s o f l i p i d s were the t a r g e t o f the photodynamic a c t i o n . When the u n s a t u r a t e d a c y l c h a i n s are o x i d i z e d , s p i n l a b e l l i n g experiments showed t h a t the membranes become more r i g i d a t a l l temperatures and t h a t the membrane phase t r a n s f o r m a t i o n temperature i n c r e a s e d from 12.7° t o 20.8°C. I n 1983, Daub and H a n g a r t e r ( 1 1 8 ) , r e p o r t e d t h a t c e r c o s p o r i n produced s u p e r o x i d e r a d i c a l s as w e l l as s i n g l e t oxygen upon exposure t o l i g h t i n the presence o f oxygen. C e r c o s p o r i n r e a c t e d w i t h c h o l e s t e r o l t o form the 5 a l p h a - h y d r o p e r o x i d e of c h o l e s t e r o l . T h i s r e a c t i o n i s s p e c i f i c f o r s i n g l e t oxygen. C e r c o s p o r i n a l s o reduced p - n i t r o b l u e t e t r a z o l i u m c h l o r i d e which i s r e a d i l y reduced by s u p e r o x i d e . Superoxide dismutase, an enzyme which r e a c t s v e r y r a p i d l y w i t h superoxide, i n h i b i t e d t h i s r e a c t i o n . In 1985, Knox and Dodge (119) i s o l a t e d h y p e r i c i n from the h a i r y St. John's wort and showed t h a t i t s e n s i t i z e d the p h o t o o x i d a t i o n of m e t h y l l i n o l e n a t e . The r e a c t i o n was i n h i b i t e d by the c a r o t e n o i d , c r o c i n . H y p e r i c i n was shown t o produce s i n g l e t oxygen due t o oxygen consumption d u r i n g the s e n s i t i z e d p h o t o o x i d a t i o n of i m i d a z o l e and a l s o due t o i n h i b i t e d r a t e s o f oxygen consumption d u r i n g the r e a c t i o n i n the presence o f deuterium o x i d e or sodium a z i d e . H y p e r i c i n a l s o caused pigment l o s s and ethane p r o d u c t i o n from pea l e a f d i s c s under l i g h t exposure. Laser Herbicides S i n c e many p e s t i c i d e s are d i s c o v e r e d as a r e s u l t of e x t e n s i v e s c r e e n i n g programs of many c a n d i d a t e c h e m i c a l s , i t i s r e a l l y not n e c e s s a r y t o understand the mechanism o f a c t i o n o f the c a n d i d a t e



14


p e s t i c i d e at f i r s t . R a t h e r , i t i s n e c e s s a r y o n l y t h a t i t be e f f e c t i v e . There does e x i s t i n the realm of the f a m i l y of p h o t o a c t i v a t e d p e s t i c i d e s , a h e r b i c i d e which was a c t u a l l y d e s i g n e d on the b a s i s of knowledge of the i n h e r e n t b i o c h e m i c a l pathways i n p l a n t s . I n 1969, E l l s w o r t h and A r o n o f f (120) i n i t i a l l y proposed t h a t c h l o r o p h y l l was b i o s y n t h e s i z e d v i a 2 a l t e r n a t e p a r a l l e l pathways i n v o l v i n g monovinyl d e r i v a t i v e s and d i v i n y l d e r i v a t i v e s . Over the next s e v e r a l y e a r s , R e b e i z and h i s coworkers s t u d i e d c h l o r o p l a s t b i o g e n e s i s i n p l a n t s (4,121). They l a t e r proposed t h a t each a l t e r nate p a r a l l e l pathway c o n t a i n e d p a r a l l e l subpathways u t i l i z i n g f u l l y e s t e r i f i e d d e r i v a t i v e s and a c i d i c d e r i v a t i v e s . They r e a l i z e d at the time t h a t the known mode of a c t i o n of no h e r b i c i d e took advantage of t h i s a s p e c t of p l a n t b i o s y n t h e s i s . If chlorophyll b i o s y n t h e s i s was used as the t a r g e t f o r the h e r b i c i d a l a c t i o n , i t would a l l o w f o r a c e r t a i n s p e c i f i c i t y . F u r t h e r , the d i v e r s i t y of c h l o r o p h y l l a b i o s y n t h e t i c pathways a l l o w e d f o r d i v e r s i t y i n design. The mechanism of a c t i o n of the h e r b i c i d e would be based on the photodynamic a c t i v i t y of the p o r p h y r i n ( t e t r a p y r r o l e ) d e r i v a t i ves which are p a r t of the c h l o r o p h y l l a b i o s y n t h e t i c scheme. T h e r e f o r e , i t would be dependent on the b i o s y n t h e s i s and accumulat i o n of the t e t r a p y r r o l e s by the sprayed p l a n t t a r g e t s . F u r t h e r , a p o s t - s p r a y p e r i o d of darkness of s e v e r a l hours would be r e q u i r e d f o r the a c c u m u l a t i o n of the t e t r a p y r r o l e s . F i n a l l y , upon exposure to l i g h t , a v e r y damaging photodynamic e f f e c t , c a t a l y z e d by the a c c u mulated t e t r a p y r r o l e s , would o c c u r which w i l l r e s u l t i n the death of the p l a n t t a r g e t . I n o r d e r to s t i m u l a t e the b i o s y n t h e s i s of t e t r a p y r r o l e s i n the p l a n t t a r g e t , d e l t a - a m i n o l e v u l i n i c a c i d and 2 , 2 - d i p y r i d y l were sprayed on cucumber s e e d l i n g s i n the dark. A f t e r 17 hours i n the d a r k , the p l a n t s were exposed t o d a y l i g h t and they s u f f e r e d e x t e n s i v e photodynamic damage. The green l e a f y t i s s u e and the h y p o c o t y l became b l e a c h e d . I n b o t h c a s e s , the t i s s u e s s u f f e r e d a severe l o s s of t u r g i d i t y , p r o b a b l y due to the development of l e a k y c e l l memmembranes, f o l l o w e d by a r a p i d and severe d e h y d r a t i o n of the tissues. P r i o r t o l i g h t exposure, some of the s e e d l i n g s were anal y z e d and i n c r e a s e d c e l l u l a r l e v e l s of t o t a l t e t r a p y r r o l e s were found to be c o n c e n t r a t i o n dependent upon the sprayed d e l t a a m i n o l e v u l i n i c a c i d and 2 , 2 - d i p y r i d y l . When o t h e r p l a n t s were t r e a t e d s i m i l a r l y , i t became apparent t h a t the d e l t a - a m i n o l e v u l i n i c a c i d and 2 , 2 ' - d i p y r i d y l induced photodynamic a c t i o n c a u s i n g 3 d i f f e r e n t types of h e r b i c i d a l responses depending upon the t a r g e t s p e c i e s . The Type I r e s p o n s e , observed i n d i c o t s such as the cucumber, i s c h a r a c t e r i z e d by a c c u m u l a t i o n s of t e t r a p y r r o l e s i n l e a f y t i s s u e s , stems, and growing p o i n t s , and r a p i d death from photodynamic a c t i o n which i s d i r e c t l y dependent upon l i g h t i n t e n s i t y . The Type I I response i s observed i n o t h e r d i c o t s such as c o t t o n , k i d ney bean, and soybean. T e t r a p y r r o l e s are accumulated i n the l e a f y t i s s u e s , but not i n the stems. Leaves t h a t accumulate the t e t r a p y r r o l e s d i e v e r y r a p i d l y w i t h i n a few hours of l i g h t exposure, but the c o t y l e d o n s , stems, and growing p o i n t s remain u n a f f e c t e d . These p l a n t s c o u l d r e c o v e r from t h i s i n i t i a l damage by p r o d u c i n g new leaves. I t was a l s o observed t h a t , i f the p l a n t s were young enough 1

1



1.

HEITZ


15

t h a t the l e a v e s were e n c l o s e d by the c o t y l e d o n s , the p l a n t s were c o m p l e t e l y u n a f f e c t e d . The Type I I I response was e x h i b i t e d o n l y by monocotyledons, such as wheat, c o r n , o a t s , or b a r l e y . I n t h i s case, the p l a n t developed s m a l l n e c r o t i c r e g i o n s when the sprayed p l a n t was exposed t o l i g h t . The s e e d l i n g s grew v i g o r o u s l y and developed i n t o h e a l t h y p l a n t s . Rebeiz and h i s coworkers have thus developed the f i r s t photodynamic h e r b i c i d e . The p o p u l a r press has a l r e a d y g i v e n t h i s c l a s s o f h e r b i c i d e another name, " l a s e r h e r b i c i d e s . " By whatever name they w i l l be c a l l e d , these h e r b i c i d e s appear t o have a p r o m i s i n g f u t u r e . The d i f f e r e n t pathways of c h l o r o p h y l l b i o s y n t h e s i s s h o u l d a l l o w a degree of f l e x i b i l i t y so t h a t p r o d u c t s developed from t h i s c l a s s o f h e r b i c i d e s w i l l be t o l e r a n t t o the crop p l a n t s and t o x i c to the weeds. In 1986, Rebeiz and Hopen were awarded a patent c o v e r i n g the l a s e r h e r b i c i d e concept ( 1 2 2 ) . Miscellaneous Materials There have been r e p o r t s of o t h e r m a t e r i a l s which may become import a n t as t h i s r e s e a r c h area d e v e l o p s . At t h i s t i m e , however, they have not a t t r a c t e d the a t t e n t i o n o f the p e s t i c i d e c l a s s e s t h a t were discussed e a r l i e r i n t h i s chapter. M a l t o t s y and F a b i a n (123-124) f i r s t found t h a t p o l y a r o m a t i c hydrocarbons were t o x i c to l a r v a e of the f r u i t f l y upon i r r a d i a t i o n w i t h UV l i g h t . The h i g h c a r c i n o g e n i c p o t e n t i a l of t h i s c l a s s of compounds has kept them from b e i n g e x p l o i t e d as much as would be expected i f t h e r e were no c a r c i n o g e n i c r i s k . Kagan and Kagan (125) addressed t h i s problem w i t h a comparative study the e f f e c t s of benzo[a]pyrene ( c a r c i n o g e n i c ) and pyrene ( n o n c a r c i n o g e n i c ) upon immature forms o f Aedes mosquitoes h e l d i n the dark or i r r a d i a t e d w i t h UV l i g h t . T h e i r r e s u l t s i n d i c a t e d t h a t c a r c i n o g e n i c i t y and p h o t o t o x i c i t y were not i n e x t r i c a b l y l i n k e d . L a t e r , Kagan e_t a l . (126) c a l l e d a t t e n t i o n t o the p o s s i b l e d e l e t e r i o u s e f f e c t s on a q u a t i c organisms of p o l y a r o m a t i c hydrocarbons i n a d v e r t a n t l y i n t r o duced i n t o the environment. Kagan e_t a l . (127) r e p o r t e d t h a t 2 , 5 - d i p h e n y l o x a z o l e , known t o workers i n s c i n t i l l a t i o n c o u n t i n g as POP, i s p h o t o t o x i c to the f i r s t i n s t a r of Aedes mosquito l a r v a e , t o c r u s t a c e a n s , and to the eggs o f f r u i t f l i e s . A s i m i l a r compound, 1 , 4 - b i s ( 5 - p h e n y l o x a z o l e 2-yl)benzene, known as POPOP, i s a l s o t o x i c , but t o a l e s s e r degree. Both can s e n s i t i z e the f o r m a t i o n of s i n g l e t oxygen. M o l e r o et^ a l . (128) r e p o r t e d a photodynamic a c t i v i t y i n r o o t t i s s u e mediated by b e r b e r i n e s u l f a t e and v i o l e t (420 nm) l i g h t . At low c o n c e n t r a t i o n s (nanomolar), r o o t growth i n h i b i t i o n was complete. The f i r s t p h o t o t o x i c l i g n a n , n o r d i h y d r o g u a i a r e t i c a c i d ( I X ) , from the l e a f r e s i n of the c r e o s o t e bush has been r e p o r t e d (Downum, K.R.; D o l e , J . ; R o d r i g u e z , E. Phytochem, i n p r e s s ) . Many more l i g n a n s are known, o c c u r r i n g i n many f a m i l i e s of p l a n t s , and they may become an i m p o r t a n t f u t u r e source of p h o t o c h e m i c a l l y a c t i v e chemicals.



OH


OH

0

OH

0

OH

Hypericin

Structure VII

OH

0

OH

0

Cercosporin

Structure VIII

Nordihydroguaiaretic acid

S t r u c t u r e IX


1. HEITZ


17


Conclusions It i s apparent that the concept of l i g h t activation of molecules to enhance b i o l o g i c a l a c t i v i t y i s a concept which i s both intriguing and currently available- Although there are applications which would not allow catalysis by l i g h t , such as the photonegative insects and most root tissue i n plants, there is a wide and diverse population of pests which do function i n the l i g h t . The f i r s t tentative steps torwards application have been taken using available synthetic chemicals and known plant materials, a l l of which were i d e n t i f i e d through general screening programs. It i s to be hoped that the next steps may follow at least i n part the approaches of Constantin Rebeiz and his coworkers i n which the toxic molecule was designed from known p r i c i p l e s of the biochemistry of the plant target. In fact, although there are no known examples thus f a r , i t would appear that the general area of photoaffinity l a b e l l i n g of enzymes may hold promise as a watershed for the development of new pesticides as s p e c i f i c targets within the pest are more completely understood. Other fundamental areas of l i g h t activation systems may s i m i l a r l y be future watersheds for pesticides based on this approach. Acknowledgments This work was supported i n f u l l by the M i s s i s s i p p i A g r i c u l t u r a l and Forestry Experiment Station. The author would l i k e to thank Mrs. Debbie Smith and Mrs. Ann Smithson for their assistance i n typing the manuscript. MAFES publication number 6524. Literature Cited 1. Heitz, J.R. In Insecticide Mode of Action; Coats, J . R . , Ed.; Academic: New York, 1982; pp. 429-457. 2. Robinson, J.R. Res. Rev. 1983, 88, 69-100. 3. Arnason, T . ; Towers, G.H.N.; Philogene, B.J.R.; Lambert, J.D.H. In Plant Resistance to Insects; Hedin, P.A., Ed.; ACS Symposium Series No. 208; American Chemical Society, Washington, DC, 1983; pp. 139-151. 4. Rebeiz, C.A.; Montazer-Zouhoor, A.; Hopen, H . J . ; Wu, S.M. Enzyme Microb. Technol. 1984, 6, 390-401. 5. Towers, G.H.N. Can. J . Bot. 1984, 62, 2900-2911. 6. Cooper, G.K.; Nitsche, C.I. Bioorg. Chem. 1985, 13, 362-374. 7. Knox, J . P . ; Dodge, A.D. Phytochem. 1985, 24, 889-896. 8. Downum, K.R. In Natural Resistance of Plants to Pests:Roles of Allelochemicals; Green, M.B.; Hedin, P.A., Eds.; ACS Symposium Series No. 296; American Chemical Society, Washington, DC, 1986; pp. 197-205. 9. Marcacci, A. Arch. Ital. Biol. 1888, 9, 2. 10. Rabb, O. Z. fur Biol. 1900, 39, 524-546. 11. Jodlbauer, A . ; von Tappeiner, H. Muench. Med. Wochenschr. 1904, 26, 1139-1141. 12. Spikes, J . D . ; Glad, B.W. Photochem. Photobiol. 1964, 3, 471-487. 13. Edwards, W.F. Text. World. 1921, 60, 1111-1113. 14. Chang, H.T. Mosquito News. 1946, 6, 122-125.


18 15. 16. 17. 18.


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