Vulnerability of Pests: Study and Exploitation for ... - ACS Publications

Chapter 6

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Vulnerability of Pests: Study and Exploitation for Safer Chemical Control Robert M. Hollingworth Department of Entomology, Purdue University, West Lafayette, IN 47907

Despite considerable improvements in their safety, the current use of pesticides causes an uncertain but disturbing level of toxicity to non-target organisms on a worldwide basis. A better knowledge of the biochemistry and physiology of pests could reduce this threat by decreasing unintended exposure to existing pesticides and by aiding in the discovery of new and more selective ones. Such knowledge can help in the discovery process in several ways — in the development of new control concepts, in the rational design of novel compounds, and by providing tools for their efficient evaluation and optimization. Scientificallybased strategies to slow the onset of resistance to such rare and valuable materials must be developed. Here, too, a better knowledge of their biochemical modes of action and of pest vulnerability and defenses will be indispensible. However these goals can be fully realized only i f there is greater investment in research into pesticidal mechanisms and responses in target and non-target species. For the last 25 years and more the use of pesticides has been a controversial and troubled subject of continuing public concern. This concern has been based on the feeling that pesticides, as presently used, may be seriously hazardous to man and to the environment. The degree to which t h i s i s true i s not c l e a r . On the one hand reasonably complete s t a t i s t i c s indicate that in the USA, which uses about 30% of the pesticide produced i n the world, less than 50 people a year are k i l l e d by accidental exposure to these materials (J^), fewer than the number k i l l e d by lightning or by insect stings. S t a t i s t i c s for other developed nations show similar mortality rates. However, worldwide estimates of the number of accidental deaths from pesticides, though very uncertain, are much higher, ranging from 5,000 to over 20,000 per year i n the early 1970 s (2), and indicating a considerable degree of misuse i n developing nations. Nor has t h i s situation c l e a r l y improved i n the l a s t decade. About 13,000 pesticide-related f

0097-6156/87/0336-0054$06.75/0 © 1987 American Chemical Society

Ragsdale and Kuhr; Pesticides ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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Vulnerability of Pests

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h o s p i t a l i z a t i o n s and 1000 d e a t h s (73% as s u i c i d e s ) were r e c o r d e d i n t h e i s l a n d o f S r i Lanka each year from 1975 t o 1980 ( J ) , and 500-650 such d e a t h s were r e c o r d e d i n t h e P h i l l i p i n e s i n 1980-1981. The h e r b i c i d e paraquat accounted f o r 93 known f a t a l i t i e s i n t h e s m a l l i s l a n d o f T r i n i d a d i n t h e s i n g l e year o f 1984 ( 4 ) . E s t i m a t e s o f t h e number o f p e o p l e i n j u r e d by p e s t i c i d e s v a r y w i d e l y b u t may be 100-times t h e a c c i d e n t a l d e a t h r a t e on a worldwide b a s i s (5.,.6). I n 1985, 2500 o c c u p a t i o n a l l y - r e l a t e d a c c i d e n t a l p e s t i c i d e p o i s o n i n g s were o f f i c i a l l y r e p o r t e d i n C a l i f o r n i a and t h e r e a l number o f i n j u r i e s may be c o n s i d e r a b l y h i g h e r . N e i t h e r i s t h e r e c o r d o f s a f e t y i n p e s t i c i d e m a n u f a c t u r i n g i n t h e USA unblemished. S e r i o u s i n j u r i e s t o workers i n v o l v e d i n m a n u f a c t u r i n g t h e i n s e c t i c i d e c h l o r d e c o n e , t h e n e m a t i c i d e d i b r o m o c h l o r o p r o p a n e (DBCP), and, p o s s i b l y , t h e i n s e c t i c i d e l e p t o p h o s have been r e p o r t e d i n t h e l a s t 15 y e a r s (£,5.). There i s p r e l i m i n a r y b u t d i s q u i e t i n g e p i d e m i o l o g i c a l e v i d e n c e t h a t f r e q u e n t u s e r s o f some h e r b i c i d e s may have an e l e v a t e d r i s k o f c o n t r a c t i n g cancer, p a r t i c u l a r l y i f standard s a f e t y p r e c a u t i o n s a r e n o t observed (e.g. 7.,]}) • The t o l l on w i l d l i f e i s u n c e r t a i n , b u t t h e r o l e o f DDT and o t h e r o r g a n o c h l o r i n e r e s i d u e s i n t h e d e c l i n e o f some r a p t o r i a l b i r d p o p u l a t i o n s i s r e a s o n a b l y w e l l e s t a b l i s h e d and t h e r e a r e r e g u l a r r e p o r t s o f t h e d e a t h o f b i r d s due t o exposure t o organophosphate i n s e c t i c i d e s such as d i a z i n o n and famphur d u r i n g normal use, among many o t h e r i n c i d e n t s and c o n c e r n s (9.). I t i s e s t i m a t e d t h a t a t l e a s t 50% o f food i t e m s i n t h e USA c o n t a i n d e t e c t a b l e p e s t i c i d e r e s i d u e s (6). A c o n s i d e r a b l e segment (77% i n a r e c e n t p o l l ) o f t h e US p o p u l a t i o n i s concerned about p e s t i c i d e r e s i d u e s i n f o o d , and, i n c r e a s i n g l y , about r e s i d u e s i n groundwater and o t h e r water t h a t i s used f o r d r i n k i n g , even though the t o x i c o l o g i c a l s i g n i f i c a n c e o f such l o w l e v e l exposure i s d u b i o u s and t h e r e s u l t s may be m a i n l y a t r i b u t e t o t h e s e n s i t i v i t y o f modern a n a l y t i c a l equipment. These and p a r a l l e l examples o f exposure t h r o u g h s p r a y d r i f t l e a d t o c o n c e r n and a r e p o o r l y t o l e r a t e d by t h o s e i n v o l v e d because o f t h e i r i n v o l u n t a r y n a t u r e , d e s p i t e (or perhaps because o f ) t h e u n c l e a r n a t u r e o f the degree and type o f h a z a r d involved. Yet p e s t i c i d e s a r e r e g a r d e d as e s s e n t i a l f o r modern s o c i e t y — as a n e c e s s a r y economic i n v e s t m e n t i n a g r i c u l t u r e and a s a v i t a l t o o l i n t h e c o n t r o l o f v e c t o r - b o r n e d i s e a s e s such a s m a l a r i a and o n c h o c e r c i a s i s . T h i s l e a d s t o a t e n s e and a d v e r s a r i a l s i t u a t i o n between proponents and opponents o f p e s t i c i d e s . The s i t u a t i o n i s a p p r o p r i a t e l y c h a r a c t e r i z e d a s "you c a n ' t l i v e w i t h them and you c a n ' t l i v e w i t h o u t them". In p a r t , t h e i n c r e a s e d p e s t i c i d a l potency o f newer compounds i s h e l p i n g t o s o l v e some o f t h e s e problems. Use r a t e s o f such compounds as t h e p h o t o s t a b l e p y r e t h r o i d and a v e r m e c t i n i n s e c t i c i d e / a c a r i c i d e s , t h e s u l f o n y l u r e a h e r b i c i d e s , and e r g o s t e r o l b i o s y n t h e s i s i n h i b i t o r s as f u n g i c i d e s a r e a few ounces o f a c t i v e i n g r e d i e n t p e r a c r e a t most. T h i s g r e a t l y r e d u c e s r e s i d u e s and d e c r e a s e s t h e chance o f a c c i d e n t a l p o i s o n i n g s . However, t h e s e compounds a r e n o t e n t i r e l y w i t h o u t h a z a r d . Many o f t h e p y r e t h r o i d s a r e e x q u i s i t e l y t o x i c t o a q u a t i c s p e c i e s , i n c l u d i n g f i s h , and some a v e r m e c t i n s have an acute o r a l t o x i c i t y t o r a t s c o n s i d e r a b l y h i g h e r than t h a t o f p a r a t h i o n . The problem, even i n t h e s e examples o f modern h i g h l y p o t e n t i n s e c t i c i d e s i s t h a t t h e y a c t on b i o c h e m i c a l t a r g e t s i t e s which a r e a l s o p r e s e n t and c r i t i c a l i n


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vertebrates i . e . the sodium channel of excitable t i s s u e s in the case of pyrethroids and GABA-ergic i n h i b i t o r y neurotransmission i n the case of avermectins. Insecticides as a class present the greatest r i s k of acute poisoning i n man and other vertebrates. They are the most d i f f i c u l t class for the discovery of new and safer materials. At the same time the usefulness of the limited range of existing compounds i s continually threatened by the development of r e s i s t a n c e . Consequently, i n s e c t i c i d e s represent both the biggest need and the greatest challenge i n devising safer pest control technologies, and special emphasis i s l a i d on insect control i n the discussion that follows. This a r t i c l e addresses the question of how we may increase the safety of pesticide use through a knowledge of pest biochemistry and physiology. Such research and knowledge can lead to improved safety both i n terms of compounds currently registered for use and, more p a r t i c u l a r l y , i n helping guide the search for new and safer materials to replace them. In t h i s , i t deals with p o s s i b i l i t i e s only. Which, i f any, of these p o s s i b i l i t e s w i l l come to f r u i t i o n depends on many factors including the l e v e l of investment in t h e i r development and p o t e n t i a l l y severe technical, economic, and regulatory constraints. Enhancing the Safety of Existing Compounds The r i s k from pesticide use depends on two factors — t o x i c i t y and exposure. Since the t o x i c i t y of an existing compound i s an innate property, i t cannot be altered, but the t o x i c i t y of the f i n a l formulation may be improved and t h i s w i l l alter the r i s k . To make the use of current compounds safer, then, i s mainly to concentrate on the reduction of exposure or the improvement of formulations and application methodologies for greater safety. Knowledge of pest biochemistry and physiology could help to achieve t h i s i n several ways. Minimizing exposure through reduced application rates or frequency. Attractants and baits for mobile pests may greatly safen the use of existing compounds by allowing the l o c a l i z e d placement of an i n s e c t i c i d e or by decreasing the o v e r a l l concentration needed i n the formulation. An excellent example i s the, use of bait formulations of the i n s e c t i c i d e , mirex, which reduced the amount of t h i s organochlorine material needed to control imported f i r e ants from about 1 kg to about 1 g per acre (10). Unfortunately, even t h i s reduction was not judged to be enough to allow the continued use of mirex for t h i s purpose because of i t s environmental s t a b i l i t y and potential carcinogenicity. Also, the use of insect pheromones to monitor insect populations and thereby reduce the frequency and improve the timing of pesticide applications i s well established (11). Applications may be reduced by as much as 50% and t h i s i n turn increases the contribution to control by b e n e f i c i a l insects, further decreasing the need for i n s e c t i c i d e treatment. In some favorable cases, pheromones can be used to a t t r a c t insects to l o c a l i z e d insecticide-treated s i t e s for safer c o n t r o l . It i s salutory to remember that early basic research on insect sex pheromones was essential for these b e n e f i t s , but was derided in some quarters as a waste of public funds on studying the sex l i v e s of insects.


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I t i s p r o b a b l e t h a t t h r o u g h a b e t t e r u n d e r s t a n d i n g o f such examples o f c h e m o r e c e p t i o n and t h e f e e d i n g b e h a v i o r o f p e s t s , new, improved a t t r a c t a n t s and b a i t f o r m u l a t i o n s c o u l d be p r o d u c e d . P a r t i c u l a r l y important i n t h i s r e g a r d a r e such i n s e c t s as t h e H e l i o t h i s complex on c o t t o n which a r e exposed t o f o l i a r i n s e c t i c i d e s o n l y b r i e f l y as t h e y h a t c h from t h e egg and m i g r a t e t o t h e c o t t o n b o l l . Much i n s e c t i c i d e i s wasted i n t r y i n g t o ensure t h a t a l e t h a l dose i s p i c k e d up by c o n t a c t d u r i n g t h i s s h o r t t i m e . The b r i e f window o f v u l n e r a b i l i t y a l s o imposes a s e v e r e l i m i t a t i o n on t h e e f f i c a c y o f such h i g h l y s a f e m a t e r i a l s as t h e B a c i l l u s t h u r i n g i e n s i s e n d o t o x i n , which must be i n g e s t e d by t h e l a r v a e t o be t o x i c . Compounds t h a t a l t e r i n s e c t b e h a v i o r i n o t h e r ways may a l s o be u s e f u l e.g. c h e m i c a l s t h a t i n c r e a s e i n s e c t locomotor a c t i v i t y s h o u l d enhance t h e uptake o f p e s t i c i d e s from t r e a t e d s u r f a c e s . Such compounds a r e a l r e a d y known among t h e formamidines i n s e c t i c i d e s and t h e i r r e l a t i v e s t h a t s t i m u l a t e octopamine r e c e p t o r s ( 1 2 ) , and f u r t h e r r e s e a r c h i n t h i s area o f pest b i o c h e m i s t r y could r e v e a l other l o c o m o t o r s t i m u l a t o r s such as p h o s p h o d i e s t e r a s e i n h i b i t o r s (1_3,JjO. A s i m i l a r p h i l o s o p h y u n d e r l i e s r e c e n t s t u d i e s showing t h a t alarm pheromones i n c r e a s e t h e m o t i l i t y o f a p h i d s and t h a t t h i s ' r e s u l t s i n the enhanced e f f i c a c y o f c o n t a c t i n s e c t i c i d e s such as p y r e t h r o i d s and organophosphates ( 1 5 ) . I t i s a l s o p r o b a b l e t h a t a more d e t a i l e d s t u d y o f t h e f a c t o r s g o v e r n i n g t h e uptake o f p e s t i c i d e s from t r e a t e d s u r f a c e s c o u l d l e a d t o improved f o r m u l a t i o n s o f h i g h e r e f f i c i e n c y and lower c o n c e n t r a t i o n . From t h e l i m i t e d d a t a a v a i l a b l e i t i s c l e a r t h a t e v e n t s on t h e s u r f a c e o f t h e l e a f o r i n s e c t and t h e type o f f o r m u l a t i o n a p p l i e d may r a d i c a l l y a f f e c t t h e performance o f h e r b i c i d e s 0 6 ) and i n s e c t i c i d e s

(J7). M o d i f i c a t i o n o f the T o x i c i t y o f P e s t i c i d e s with Formulation Additives. P o t e n t i a l l y t h e use o f a d d i t i v e s t o modulate t h e t o x i c i t y of p e s t i c i d e s could lead to a considerable increase i n t h e i r s a f e t y t o n o n - t a r g e t s p e c i e s , man i n c l u d e d . T h i s c o n c e p t has been w e l l e x p l o r e d and e x p l o i t e d i n adding s a f e n e r s t o c e r t a i n h e r b i c i d e s such as t h e t h i o c a r b a m a t e s . These compounds s t i m u l a t e d e f e n s i v e m e t a b o l i c r e a c t i o n s i n t h e c r o p s p e c i e s b u t n o t i n weeds ( 1 8 , 1 . 9 ) . T h i s p r i n c i p l e has a l s o been a p p l i e d t o v e r t e b r a t e s , b u t o n l y t o a v e r y l i m i t e d d e g r e e . Under some c i r c u m s t a n c e s t h e t h i o c a r b a m a t e r i c e h e r b i c i d e , m o l i n a t e , may show t o x i c i t y t o c a r p i n nearby ponds. Based on a knowledge o f t h e s a f e n e r s t h a t a r e a c t i v e i n p l a n t s , a compound was d i s c o v e r e d t h a t , when a p p l i e d w i t h m o l i n a t e , a c t e d as an a n t i d o t e / s a f e n e r f o r t h e c a r p ( 2 0 ) . L i t t l e e f f o r t , e i t h e r t h e o r e t i c a l o r e m p i r i c a l , seems t o have gone i n t o d e v e l o p i n g o t h e r such examples. Since " s a f e n i n g " i n v o l v e s t r a n s i e n t l y changing the b i o c h e m i s t r y o f n o n - t a r g e t s p e c i e s , t h e a l t e r n a t i v e s t r a t e g y o f making t h e p e s t i c i d e s e l e c t i v e l y more a c t i v e w i t h s y n e r g i s t s t h a t a l t e r t h e p e s t ' s b i o c h e m i s t r y may be more a p p e a l i n g . Numerous examples o f s y n e r g i s m w i t h a l l t y p e s o f p e s t i c i d e s a r e known, but t h e p r e s e n t a p p l i c a t i o n s o f t h e p r i n c i p l e a r e l i m i t e d by s e v e r a l f a c t o r s i n c l u d i n g a l a c k o f s e l e c t i v i t y f o r non-target species i n the s y n e r g i s t i c p r o c e s s (J9., 21_). The d i s c o v e r y and a p p l i c a t i o n o f p e s t - s p e c i f i c s y n e r g i s t s i s a r e a l i s t i c g o a l which, i f e x p l o i t e d , c o u l d l e a d t o c o n s i d e r a b l y reduced f o r m u l a t i o n and a p p l i c a t i o n r a t e s


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and t h u s t o a h i g h e r l e v e l o f s a f e t y t o t h e a p p l i c a t o r and e n v i r o n m e n t . One compound f o r which such an approach i s j u s t i f i e d i s the h e r b i c i d e paraquat which, based on t h e known number o f human p o i s o n i n g s , i s one o f the most hazardous p e s t i c i d e s c u r r e n t l y i n u s e . In b o t h p l a n t s and a n i m a l s i t s t o x i c a c t i o n s r e s u l t from t h e c a t a l y t i c g e n e r a t i o n o f r e a c t i v e forms o f oxygen i n t h e t i s s u e s . S u c c e s s i n t h e use o f copper and z i n c c h e l a t o r s t o s y n e r g i z e i t s t o x i c i t y by i n h i b i t i n g t h e a n t i o x i d a n t enzymes ( e . g . s u p e r o x i d e d i s m u t a s e ) t h a t tend t o p r o t e c t t h e p l a n t i s t h e r e f o r e p r o m i s i n g (22). S y n e r g i s t s t h a t a r e r e l a t i v e l y s e l e c t i v e between p l a n t s and a n i m a l s i n t h i s r e g a r d would be p a r t i c u l a r l y v a l u a b l e . Such s y n e r g i s t s , s e l e c t i v e o r n o t , c o u l d n o t have been d e v i s e d w i t h o u t a t h o r o u g h knowledge o f the mechanism o f a c t i o n and p h y s i o l o g i c a l r e s p o n s e s t o paraquat i n p l a n t s . The c o n c e p t o f s e l e c t i v e synergism o f p e s t i c i d e s has c o n s i d e r a b l e p o t e n t i a l b u t , as y e t , has h a r d l y been exploited. S t r a t e g i e s t o Overcome P e s t i c i d e

Resistance

The development o f r e s i s t a n c e i s a p r o c e s s which i n e x o r a b l y e l i m i n a t e s e x i s t i n g p e s t i c i d e s from t h e market, s a f e and dangerous alike. Even i f t h e y a r e n o t e l i m i n a t e d , a p p l i c a t i o n r a t e s may have t o be r a i s e d a s r e s i s t a n c e d e v e l o p s l e a d i n g t o enhanced l e v e l s o f exposure f o r n o n - t a r g e t o r g a n i s m s . Even w i t h h e r b i c i d e s , r e s i s t a n c e i s now s t a r t i n g t o become a p r a c t i c a l problem; i t i s an e s t a b l i s h e d one f o r i n s e c t i c i d e s and t h e modern s e l e c t i v e f u n g i c i d e s . It i s p r o b a b l y a worse t h r e a t t o s a f e r compounds s i n c e t h e s e o f t e n a r e s e l e c t i v e l y t o x i c by a t t a c k i n g a s i n g l e enzyme o r r e c e p t o r p e c u l i a r t o t h e p e s t group. A s i n g l e m u t a t i o n a t t h i s s i t e may then r e n d e r the t a r g e t i n s e n s i t i v e and t h e pest h i g h l y r e s i s t a n t . Compounds a c t i n g a t m u l t i p l e e s s e n t i a l s i t e s a r e l e s s open t o t h e development o f t a r g e t s i t e r e s i s t a n c e , b u t by the same token a r e l e s s l i k e l y t o be s e l e c t i v e i n t h e i r t o x i c a c t i o n s . P r e s e r v i n g s a f e p e s t i c i d e s by s l o w i n g , p r e v e n t i n g , o r r e v e r s i n g t h e o n s e t o f r e s i s t a n c e depends a b s o l u t e l y on u n d e r s t a n d i n g t h e b i o c h e m i s t r y and p h y s i o l o g y o f t h e pest and t h e b i o c h e m i c a l and p o p u l a t i o n g e n e t i c s o f t h e r e s i s t a n c e process. A f t e r many y e a r s o f r e l a t i v e p a s s i v i t y i n t h e f a c e o f r e s i s t a n c e , a more a g g r e s s i v e c o n c e p t o f " r e s i s t a n c e management" has d e v e l o p e d c o n s i d e r a b l e momentum i n t h e l a s t decade a s i t has become c l e a r t h a t new p e s t i c i d e s w i l l n o t be r e a d i l y a v a i l a b l e t o r e p l a c e those l o s t t o r e s i s t a n c e . T h i s approach i s reviewed elsewhere much more f u l l y than i s p o s s i b l e h e r e (21,23-26). There a r e s e v e r a l c r i t i c a l a r e a s o f r e s i s t a n c e management where a knowledge o f p e s t b i o c h e m i s t r y and p h y s i o l o g y i s e s s e n t i a l : 1.

D e f i n i t i o n o f e x i s t i n g and p o t e n t i a l r e s i s t a n c e mechanisms and t h e i r g e n e t i c b a s i s .

2.

Development o f r a p i d , s i m p l e , and cheap methods t o m o n i t o r r e s i s t a n c e l e v e l s and mechanisms o c c u r r i n g a t a low l e v e l i n the f i e l d .

3.

Development o f c h e m i c a l s t r a t e g i e s t o a l l e v i a t e o r p r e v e n t resistance.


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Resistance to pesticides arises primarily through changes i n the s e n s i t i v i t y of the s i t e of action or in the metabolism of the pesticide (25,27,28). Many pesticides are activated metabolically. While i t i s t h e o r e t i c a l l y possible to generate resistance through reduced a c t i v a t i o n , i t seems much more common to observe increased d e t o x i f i c a t i o n i n resistant s t r a i n s . In some cases decreased uptake or enhanced excretion also contribute. It i s an obvious prerequisite for any type of s c i e n t i f i c a l l y - b a s e d attempt to combat resistance that the resistance mechanism and i t s genetic basis must be defined. There i s a clear need for rapid, simple and cheap methods to monitor the status of resistance mechanisms and l e v e l s while they are s t i l l at a low incidence in f i e l d populations. If t h i s can be achieved, early warning of changes in resistance gene frequency within the population w i l l be possible. This should allow a s h i f t to alternative control measures before the gene frequency and associated development of a supportive genotype progress to a stage which results i n the i r r e v e r s i b l e loss of a desirable p e s t i c i d e . The same survey methods w i l l also be essential for monitoring the success of these alternative strategies. To be successful, a l t e r a t i o n s i n gene frequency need to be detected when no more than a few percent of the population has the resistance gene. This demands a high degree of s e n s i t i v i t y and r e l i a b i l i t y at the population l e v e l that i s not possible with t y p i c a l bioassay methods. However biochemical tests for the presence of the resistance gene in individuals in the population may meet these requirements. Some examples of approaches to such tests are already available e.g. assays for esterase l e v e l s in individual l e a f - and planthoppers, mosquitoes, and aphids that could be run under f i e l d conditions or in l o c a l laboratories in the search for individuals with an elevated a c t i v i t y that provides resistance to organophosphates and other esters (29,30). Other simple tests for enzymological markers of resistance seem feasible based on the c a t a l y t i c a c t i v i t y of the resistance s i t e such as changes i n the s e n s i t i v i t y of a c e t y l c h o l i n esterase (AChE) to i n h i b i t o r s (2£), mixed function oxidase (MFO) a c t i v i t y , and glutathione transferase a c t i v i t y . In the herbicide area, a f i e l d test for altered s e n s i t i v i t y to photosynthesis i n h i b i t o r s has been described (21). However, developing s p e c i f i c diagnostic methods for some other t r a i t s involving changed target s i t e s such as the kdr resistance mechanism for pyrethroids w i l l be more challenging. With the p u r i f i c a t i o n of the changed enzymes or receptors responsible for resistance has come the p o s s i b i l i t y of gene cloning and the development of highly sensitive and s p e c i f i c diagnostic tests based on immunological methods such as ELISA assays (32,33). The third element in resistance management i s the development of strategies to a l l e v i a t e resistance. Several p o s s i b i l i t i e s can be envisioned (Table I ) . The various approaches in Table I and their d i f f i c u l t i e s have a l l been discussed previously (21,25,26), and some have been exploited on occasion by an e s s e n t i a l l y empirical approach. The i n t e l l i g e n t use of mixtures or alternations of d i s s i m i l a r materials could be viewed as an attempt to gain the resistance-delaying advantages of a pesticide with multiple sites of action. Although widely used in fungicidal treatments, the r a t i o n a l development of t h i s approach i s s t i l l in i t s infancy. Besides the desired suppression of the onset of resistance, i t also c a r r i e s the


60


h a z a r d o f t h e r a p i d development o f r e s i s t a n c e t o b o t h t y p e s o f p e s t i c i d e , e.g. b y t h e s e l e c t i o n f o r a d e t o x i f i c a t i o n mechanism t h a t a f f e c t s b o t h compounds. P r e d i c t i o n s o f which o f t h e s e outcome may o c c u r a r e s t i l l hazardous because o f our l a c k o f b a s i c knowledge. A l l o f t h e methods i n T a b l e I w i l l be more a c c e s s i b l e and p r e d i c t a b l e o n l y a s our u n d e r s t a n d i n g o f p e s t b i o c h e m i s t r y and p h y s i o l o g y increases.

Table I.

Some Chemical S t r a t e g i e s t o A l l e v i a t e

1. Use o f p e s t i c i d e s w i t h m u l t i p l e

Resistance.

sites of action.

2.

Use o f m i x t u r e s o f compounds w i t h d i s s i m i l a r modes o f a c t i o n that lack cross-resistance p o t e n t i a l .

3.

A l t e r n a t i o n s and r o t a t i o n s o f such d i s s i m i l a r compounds.

4.

Use o f a d d i t i v e s t h a t a n t a g o n i z e t h e a d a p t i v e v a l u e o f t h e r e s i s t a n c e mechanism.

5.

Use o f compounds t h a t d i s p l a y cross-resistance.

negatively-correlated

In a number o f i n s t a n c e s t h e use o f s y n e r g i s t s t o a n t a g o n i z e the a d a p t i v e advantage o f a m e t a b o l i c r e s i s t a n c e mechanism has been t r i e d , b u t w i t h v a r y i n g r e s u l t s ( 3 4 . ) . One s u c c e s s f u l example i s t h e use o f e s t e r a s e i n h i b i t o r s t o p r e v e n t r e s i s t a n c e t o organophosphates i n l e a f - and p l a n t h o p p e r s i n Japan ( 3 5 ) . Once t h e mechanism o f r e s i s t a n c e i s understood a t t h e biochemical l e v e l , s t r a t e g i e s t o combat o t h e r t y p e s o f r e s i s t a n c e c a n be e n v i s i o n e d e.g. t h e use o f m i t o c h o n d r i a l p o i s o n s t o p r e v e n t t h e energy-dependent e x p u l s i o n o f s t e r o l b i o s y n t h e s i s i n h i b i t o r s by some f u n g i ( 3 6 ) . It i s harder, but not impossible, t o e n v i s i o n s y n e r g i s t s that a c t t o oppose a l o s s o f s e n s i t i v i t y a t t h e s i t e o f a c t i o n . However, i n these cases, other s t r a t e g i e s are a v a i l a b l e since a b i o l o g i c a l l y e s s e n t i a l t a r g e t s i t e (enzyme, r e c e p t o r ) g e n e r a l l y cannot d i s a p p e a r but i s o n l y changed somewhat i n i t s p r o p e r t i e s i n t h e mutant form. T h i s i m p l i e s t h a t i n some c a s e s , o t h e r compounds may be found t h a t a r e e f f e c t i v e a g a i n s t t h e changed s i t e and which t h e r e f o r e a r e t o x i c t o t h e r e s i s t a n t p e s t . T h i s s i t u a t i o n , which should be p o w e r f u l i n p r e v e n t i n g or even r e v e r s i n g t h e e f f e c t s o f r e s i s t a n c e , i s an example of n e g a t i v e l y - c o r r e l a t e d c r o s s - r e s i s t a n c e i . e . the higher t o x i c i t y o f a compound t o t h e r e s i s t a n t than t h e s u s c e p t i b l e s t r a i n . Such compounds have been been d i s c o v e r e d and a r e a l r e a d y b e i n g u t i l i z e d i n a l i m i t e d number o f s i t u a t i o n s . F o r example, i t has been found t h a t r e s i s t a n c e t o t y p i c a l carbamate i n s e c t i c i d e s i n t h e g r e e n r i c e l e a f h o p p e r o f t e n i n v o l v e s t h e development o f a mutant form o f AChE which i s i n s e n s i t i v e t o i n h i b i t i o n by N n n e t h y l c a r b a m a t e s . However, a s i t e on t h e changed AChE i s now s e n s i t i v e t o N^propylcarbamates which are r e l a t i v e l y i n a c t i v e on t h e n a t i v e enzyme ( 3 7 ; F i g u r e 1 ) . Presumably t h i s s i t e has undergone a change i n topography t h a t a l l o w s


6.

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METHYL

61


ETHYL

n-PROPYL

n-BUTYL

F i g u r e 1. Comparative i n h i b i t i o n ( p I 5 0 ) o f a c e t y l c h o l i n e s t e r a s e from s u s c e p t i b l e (S) and two r e s i s t a n t (RN-N and RN-4) s t r a i n s o f green r i c e l e a f h o p p e r by m - t o l y l JJ-alkylcarbamates i n which t h e ] J - a l k y l group v a r i e s from methyl t o n - b u t y l . Reproduced w i t h p e r m i s s i o n from Ref. 37. C o p y r i g h t 1983 Plenum P r e s s .


62

PESTICIDES: MINIMIZING THE

RISKS

greater bulk i n t h i s ^J-alkyl part of the i n h i b i t o r molecule. The difference in s e n s i t i v i t i e s of the forms of AChE to i n h i b i t i o n i s paralleled by differences in the t o x i c i t y of ^-methyl and Jtf-propylcarbamates to susceptible and r e s i s t a n t leafhoppers (27). More recently a comparable enhanced i n h i b i t i o n in r e s i s t a n t s t r a i n s has been observed with aryloxadiazolone anticholinesterases (38). A second promising example i s the discovery that some natural and synthetic isobutylamides are s e l e c t i v e l y toxic against houseflies that carry the super-kdr resistance t r a i t (39). This gene causes an a l t e r a t i o n in the s e n s i t i v i t y of the s i t e of action for DDT and pyrethroids and i s a major threat to the continued e f f i c a c y of synthetic pyrethroids i n many of their applications. Turning to the fungicide area, i t has been shown that resistance to benzimidazole fungicides often r e s u l t s from a change i n the benzimidazole binding s i t e on beta-tubulin or from enhanced s t a b i l i t y of the microtubules. Some h e r b i c i d a l N-phenylcarbamates which a f f e c t microtubule functioning in plants have been found to be s p e c i f i c a l l y toxic to benzimidazole-resistant s t r a i n s of fungi (28). Field tests are currently being conducted with one analog that i s not h e r b i c i d a l , S-32165 (diethofencarb; isopropyl 3,4-diethoxyphenylcarbamate), to evaluate i t s use as a "resistance breaker" (40). Another example of negatively-correlated cross-resistance among fungicides involving phosphorothiolates and phosphoramidates has been described (41). As information on the molecular architecture and mechanisms of pesticide s i t e s of action becomes more generally available and the nature and e f f e c t of mutations on their s e n s i t i v i t y to pesticides i s defined, i t should become possible in some cases to design agents that s p e c i f i c a l l y i n t e r f e r e with the altered s i t e of the r e s i s t a n t forms. The same l i n e of reasoning suggests that the design of s p e c i f i c and s e l e c t i v e synergists to block i n d i v i d u a l metabolic resistance mechanisms may eventually be possible. New

and

Safer Pesticides - Exploiting Pest V u l n e r a b i l i t y

The defects of current pesticides, regulatory actions r e s u l t i n g from them, the natural desire of chemical companies to discover new and better compounds than their competitors, and the regular loss of pesticide e f f i c a c y due to resistance and changes i n a g r i c u l t u r a l technology a l l provide impetus to the search for new compounds. However, increased research and regulatory demands are expensive and the d i f f i c u l t y of finding molecules with c l e a r l y superior properties to those of existing compounds has made the discovery of new compounds with improved safety c h a r a c t e r i s t i c s an increasingly c o s t l y and rare occurrence. It has been pointed out that the peak of innovation in the introduction of new pesticides was seen i n the l a t e 1960 s at about 20 compounds per year. Since then i t has declined p r e c i p i t o u s l y (25,42,43). Recently the success rate i s estimated to be one compound commercialized for every 15,000 to 20,000 synthesized with the t o t a l cost of developing t h i s single new compound being anywhere from $20 m i l l i o n (25) to $45 m i l l i o n (44). Pesticide discovery i s a game of roulette on a wheel with many thousands of numbers. To stay in t h i s game long enough to h i t winners and recoup investment i s extremely expensive. An increasing number of companies have decided that they cannot afford to play and have cashed i n their chips. In i t s e l f , t h i s decreases our chances of discovering safer p e s t i c i d e s . f



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63

On the b a s i s o f the o l d a d v i c e t o know t h y enemy, can s t u d i e s o f pest p h y s i o l o g y and b i o c h e m i s t r y a i d i n t h e s t r u g g l e t o f i n d new and s a f e r c h e m i c a l s f o r pest c o n t r o l ? The answer i s by no means c l e a r , but i t i s obvious t h a t the f i g u r e s f o r success u s i n g p a s t methods o f b l i n d s c r e e n i n g and analog s y n t h e s i s a r e i n c r e a s i n g l y u n f a v o r a b l e and uneconomical. The o n l y a l t e r n a t i v e i n s i g h t i s t o attempt t o a p p l y t h e growing knowledge o f p e s t b i o c h e m i s t r y and p h y s i o l o g y t o d e f i n e s p e c i f i c v u l n e r a b i l i t i e s i n t h e pest and t h u s t o decrease t h e odds i n our s e a r c h f o r b e t t e r m a t e r i a l s . T h i s p r o s p e c t has been d i s c u s s e d r e c e n t l y by a number o f a u t h o r s (19,20,43,45-48). A l t h o u g h the t o p i c o f t h e g e n e t i c e n g i n e e r i n g a s a means o f p e s t c o n t r o l i s beyond t h e scope o f t h i s c h a p t e r , i t e q u a l l y depends on such a knowledge o f t h e enemy. F o r t u n a t e l y i t i s l i k e l y t h a t r e s e a r c h on p e s t b i o c h e m i s t r y and p h y s i o l o g y can h e l p improve our chances o f f i n d i n g n o v e l compounds, p a r t i c u l a r l y s a f e r ones, a t s e v e r a l d i f f e r e n t l e v e l s . Improved B i o e v a l u a t i o n and O p t i m i z a t i o n o f Leads. At t h e s i m p l e s t l e v e l , such i n f o r m a t i o n p r o v i d e s improved methods f o r s c r e e n i n g and o p t i m i z i n g the a c t i v i t y i n a new s e r i e s o f p o t e n t i a l p e s t i c i d e s , and t o some e x t e n t i n the d i s c o v e r y o f such l e a d s a l s o . Compounds w i t h n o v e l c h e m i s t r y and m a r g i n a l b i o l o g i c a l a c t i v i t y appear more o r l e s s f r e q u e n t l y i n s c r e e n i n g , and i t i s i m p o r t a n t t o d e c i d e whether t h e s e r e p r e s e n t i m p o r t a n t l e a d s f o r new p r o d u c t s o r dead ends. Low a c t i v i t y may be due t o any one o f s e v e r a l f a c t o r s s i n g l y o r i n c o m b i n a t i o n . S t u d i e s on pest p h y s i o l o g y and b i o c h e m i s t r y p r o v i d e a b a t t e r y o f t o o l s t o a i d i n making t h i s d e c i s i o n . Poor uptake can o f t e n be d e t e c t e d i f the compound i s i n j e c t e d i n t o t h e organism or a p p l i e d i n a s p e c i a l s o l v e n t such a s d i m e t h y l s u l f o x i d e , t o o - r a p i d metabolism i s i n d i c a t e d by t h e s y n e r g i s t i c e f f e c t o f c o - a p p l i c a t i o n w i t h s p e c i f i c enzyme i n h i b i t o r s , and l o w i n t r i n s i c a c t i v i t y can be d e t e c t e d by s p e c i f i c assays o f the a c t i o n on t h e t a r g e t s i t e , when t h i s i s known. T h i s modest g o a l o f i n t e g r a t i n g our p r e s e n t knowledge o f the pest and i t s defenses i n t o t h e d i s c o v e r y and o p t i m i z a t i o n p r o c e s s sounds s i m p l e b u t even now i s n o t always a c h i e v e d . A good example o f how t h i s p r o c e s s can work i s p r o v i d e d by t h e d i s c o v e r y o f t h e n i t r o m e t h y l e n e h e t e r o c y c l i c (NMH) i n s e c t i c i d e s a t S h e l l Development Co., Modesto, CA. The i n i t i a l NMH's were o b t a i n e d from an o u t s i d e source as random s c r e e n i n g i t e m s . In t h e s c r e e n i t was found t h a t one NMH had 1% o f the a c t i v i t y o f p a r a t h i o n against h o u s e f l i e s . This i s not a very impressive l e v e l o f a c t i v i t y , but s i n c e t h e NMH s were new c h e m i s t r y , t h e r e was a r a p i d f o l l o w up i n which the compound was i n j e c t e d i n t o h o u s e f l i e s t o s u b v e r t p e n e t r a t i o n b a r r i e r s and i n t h e presence o f the s y n e r g i s t , sesamex, to i n h i b i t o x i d a t i v e degradation. This increased i t s a c t i v i t y against the h o u s e f l y t o 50% o f p a r a t h i o n s a c t i v i t y , which encouraged f u r t h e r a n a l o g s y n t h e s i s . At t h e same t i m e , mode o f a c t i o n s t u d i e s were i n i t i a t e d . The s t r o n g e x c i t a t o r y symptoms i n v i v o suggested t h a t t h e nervous system was i n v o l v e d . The compound was a p p l i e d t o t h e 6 t h abdominal g a n g l i o n p r e p a r a t i o n from the American cockroach where a m a s s i v e i n c r e a s e i n nerve a c t i v i t y was observed f o l l o w e d by a b l o c k o f n e u r o t r a n s m i s s i o n ( F i g u r e 2 ) . These o b s e r v a t i o n s , supplemented by f u r t h e r study based on t h e known p r o p e r t i e s o f t h i s p r e p a r a t i o n , suggested t h a t t h e NMH s a c t e d t o s t i m u l a t e a c e t y l c h o l i n e r e c e p t o r s f

f

1


64


CHN0

Y

(J H 2

2

5

Compound 13

A

B

Btocfctd F i g u r e 2. The development o f spontaneous g i a n t f i b e r d i s c h a r g e s (A) and t h e subsequent b l o c k o f t h e r e s p o n s e evoked by e l e c t r i c a l s t i m u l a t i o n (B) i n t h e c e n t r a l nervous system o f t h e American c o c k r o a c h t r e a t e d w i t h a 3 uM s o l u t i o n o f NMH compound 13 i l l u s t r a t e d . Reproduced w i t h p e r m i s s i o n from Ref. 49. C o p y r i g h t 1984 Academic P r e s s .


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65

(49). T h i s i s a t a r g e t s i t e f o r v e r y few c u r r e n t i n s e c t i c i d e s , and r e s i s t a n c e t o compounds a c t i n g t h e r e has not been d e v e l o p e d . T h i s d i s c o v e r y gave a d d i t i o n a l momentum t o the s y n t h e s i s program. E v e n t u a l l y compounds w i t h a c t i v i t y comparable t o the s y n t h e t i c p y r e t h r o i d s were d i s c o v e r e d (50) and i n v e s t i g a t i o n s i n t h i s a r e a continue. The d i s c o v e r y o f a mechanism o f a c t i o n i s not always so r e a d i l y a c h i e v e d , and may be v e r y c h a l l e n g i n g i f i t i s c o m p l e t e l y n o v e l . However, here p e s t i c i d e r e s i s t a n c e can o f f e r some n o t a b l e advantages. S t r a i n s o f p e s t s w i t h c a r e f u l l y d e f i n e d s i n g l e r e s i s t a n c e mechanisms i n v o l v i n g i n s e n s i t i v e s i t e s o f a c t i o n , such as p y r e t h r o i d r e s i s t a n t i n s e c t s w i t h the kdr gene or p l a n t s w i t h an a l t e r e d t r i a z i n e - b i n d i n g s i t e i n the c h l o r o p l a s t s , c a n , by t h e i r r e l a t i v e r e s i s t a n c e t o a new compound, be a u s e f u l and v e r y r a p i d d i a g n o s t i c a i d f o r e v a l u a t i n g i t s mode o f a c t i o n . Comparative s t u d i e s w i t h s t r a i n s h a v i n g e l e v a t e d l e v e l s o f such d e f e n s i v e enzymes as MFO and GSH t r a n s f e r a s e c o u l d h e l p t o d e f i n e the s u s c e p t i b i l i t y t o metabolism as a l i m i t i n g f a c t o r i n new compounds, and i n d i c a t e the p o t e n t i a l f o r r e s i s t a n c e t o them by e x i s t i n g g e n e t i c mechanisms. Rapid i n v i t r o t e s t s f o r a c t i o n s a t s e n s i t i v e t a r g e t s i t e s , such as enzyme and r e c e p t o r b i n d i n g a s s a y s , a r e needed t o h e l p i n d e f i n i n g modes o f a c t i o n e a r l y i n the development o f a new s e r i e s , and i n g u i d i n g the c h e m i s t as changes i n i n s t r i n s i c a c t i v i t y o c c u r d u r i n g i t s o p t i m i z a t i o n . C u r r e n t l y we do have good a s s a y s f o r some enzymes and f o r ACh r e c e p t o r s i n i n s e c t s , b u t methods f o r o t h e r s i t e s need t o be d e v e l o p e d f o r r o u t i n e use. T h i s i s i n s t r o n g c o n t r a s t t o the p h a r m a c e u t i c a l i n d u s t r y where i n v i t r o t e s t s a r e advanced and w i d e l y used e.g. b a t t e r i e s o f r e c e p t o r b i n d i n g a s s a y s t o h e l p d e f i n e not o n l y the p r i m a r y a c t i v i t y o f a new compound but a l s o i t s e f f e c t s on o t h e r r e c e p t o r t y p e s as an i n d i c a t o r o f p o t e n t i a l s i d e - e f f e c t s . Further b a s i c research i s e s s e n t i a l to provide t h i s c a p a b i l i t y with p e s t i c i d e s . T h i s may e v e n t u a l l y a l l o w us t o use i n v i t r o a s s a y s t o a i d i n p r e d i c t i n g at an e a r l y s t a g e o f development n o t o n l y t h e i n t r i n s i c a c t i v i t y o f compounds, but a l s o t h e i r p o t e n t i a l s i d e e f f e c t s such as impact on i m p o r t a n t n o n - t a r g e t s p e c i e s i n c l u d i n g b e n e f i c i a l i n s e c t s and v e r t e b r a t e s . In some c a s e s , such automated b i o c h e m i c a l methods may h e l p i n t h e d i s c o v e r y p r o c e s s s i n c e many more samples can be r u n t h r o u g h a s s a y s f o r AChE i n h i b i t o r s , r e c e p t o r b i n d i n g , H i l l r e a c t i o n i n h i b i t o r s , and metabolism i n h i b i t o r s as p o t e n t i a l s y n e r g i s t s than can be put t h r o u g h a normal b i o l o g i c a l s c r e e n i n the same time p e r i o d . As l o n g as such b i o c h e m i c a l s c r e e n s are r e l e v a n t f o r t h e b i o l o g i c a l a c t i v i t y d e s i r e d (43), and a r e not meant t o r e p l a c e whole animal s c r e e n i n g , but s i m p l y t o augment i t and p r o v i d e i n f o r m a t i o n on potentially interesting i n t r i n s i c a c t i v i t y for further evaluation, t h e i r u t i l i t y may be c o n s i d e r a b l e . For example, such automated s c r e e n i n g w i t h AChE as the t a r g e t r e s u l t e d i n the d i s c o v e r y o f a n o v e l s e r i e s o f i n h i b i t o r s (5J[), a l t h o u g h u n f o r t u n a t e l y w i t h s e l e c t i v e a c t i v i t y a g a i n s t mammalian forms o f the enzyme. D i s c o v e r y o f t h e mode and s i t e o f a c t i o n o f e x i s t i n g compounds. This i s a second way i n which s t u d i e s on p e s t b i o c h e m i s t r y and p h y s i o l o g y can a i d i n t h e d i s c o v e r y o f new and s a f e r compounds. The d i s c o v e r y o f the s i t e and mechanism o f a c t i o n o f a group o f p e s t i c i d e s i n e v i t a b l y s t i m u l a t e s new t h i n k i n g , p a r t i c u l a r l y i f the s i t e i s a n o v e l one.


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A r e c e n t example from the a r e a o f h e r b i c i d e s i l l u s t r a t e s t h i s p o s s i b i l i t y v e r y w e l l . Two r e c e n t new groups o f h e r b i c i d e s w i t h v e r y h i g h commercial p o t e n t i a l are the s u l f o n y l u r e a s and i m i d a z o l i n o n e s . I t was found t h a t , though q u i t e d i f f e r e n t s t r u c t u r a l l y , t h e s e compounds i n h i b i t the same enzyme i n p l a n t s , a c e t o h y d r o x y a c i d s y n t h a s e (AHAS), which i s e s s e n t i a l f o r the s y n t h e s i s o f the branched c h a i n amino a c i d s v a l i n e , l e u c i n e and i s o l e u c i n e . The mechanism o f i n h i b i t i o n i s s t i l l under s t u d y , but p r o b a b l y b o t h groups o f compounds a c t on a r e g u l a t o r y s i t e r a t h e r than on the a c t i v e s i t e o f the enzyme ( 5 2 ) . V a l i n e i t s e l f i s known t o e x e r t feedback i n h i b i t i o n on the a c t i v i t y o f some forms o f AHAS. Using t h i s i n f o r m a t i o n on the p o t e n t i a l s i t e and mode o f a c t i o n , Huppatz and C a s i d a ( 5 ^ , F i g u r e 3) noted t h a t the i m i d a z o l i n o n e s c o n t a i n a 2 - m e t h y l v a l i n e s u b s t r u c t u r e and p o s t u l a t e d t h a t o t h e r v a l i n e d e r i v a t i v e s might t h e r e f o r e a l s o i n t e r a c t a t the r e g u l a t o r y s i t e t o i n h i b i t AHAS a c t i v i t y . A s e r i e s o f v a l i n e a n a l o g s were s y n t h e s i z e d , and the most a c t i v e one, N - p h t h a l o y l - L - v a l i n e a n i l i d e , was found t o i n h i b i t AHAS and p l a n t growth i n t h e m i c r o m o l a r r a n g e . I t must be s t r e s s e d t h a t the c o n t r i b u t i o n s o f p e s t i c i d e mode o f a c t i o n s t u d i e s are not l i m i t e d t o t h e i r undoubted p r a c t i c a l s i g n i f i c a n c e i n p e s t i c i d e d i s c o v e r y . The d e f i n i t i o n o f modes o f a c t i o n i n p e s t s g e n e r a l l y has c o n s i d e r a b l e s i g n i f i c a n c e i n the s a f e t y e v a l u a t i o n s o f a p e s t i c i d e . F u r t h e r , the e l u c i d a t i o n o f a new mechanism o f a c t i o n may produce r e m a r k a b l e b e n e f i t s f o r b a s i c b i o l o g i c a l research. I t frequently stimulates research i n that f i e l d , o f t e n g e n e r a t e s new i n s i g h t s i n t o e s s e n t i a l b i o l o g i c a l p r o c e s s e s , and p r o v i d e s i n d i s p e n s i b l e t o o l s f o r t h e i r s t u d y . I t would be s a f e t o say t h a t many o f t h e most s i g n i f i c a n t advances i n modern b i o l o g y depended on the use o f n a t u r a l and man-made p o i s o n s a s p r o b e s . An even h i g h e r degree o f s o p h i s t i c a t i o n i n p e s t i c i d e d i s c o v e r y i s now w i t h i n s i g h t . The i n c r e a s i n g l y d e t a i l e d u n d e r s t a n d i n g o f enzyme mechanisms sometimes a l l o w s t h e d e s i g n o f i n h i b i t o r s t h a t i n t e r a c t v e r y s t r o n g l y o r i r r e v e r s i b l y a s w e l l as s p e c i f i c a l l y w i t h them e.g. t r a n s i t i o n s t a t e a n a l o g i n h i b i t o r s and s u i c i d e s u b s t r a t e s (48). Remarkable advances i n b i o c h e m i c a l and m o l e c u l a r g e n e t i c t e c h n i q u e s and X-ray c r y s t a l l o g r a p h y a l l o w t h e amino a c i d sequences and p r e d i c t i o n o f 3 - d i m e n s i o n a l s t r u c t u r e s o f i m p o r t a n t t a r g e t s i t e s to be g e n e r a t e d w i t h i n c r e a s i n g f a c i l i t y . The c o m p u t e r - a s s i s t e d d e f i n i t i o n o f t h e s t r u c t u r e and c h e m i s t r y o f b i n d i n g domains o f p o t e n t i a l t a r g e t r e c e p t o r s and enzymes and o f t h e i r i n t e r a c t i o n s w i t h l i g a n d s i s a l r e a d y b e i n g p r a c t i c e d ( j Q , 5 ^ , 5 5 ) . T h i s i s d i s c u s s e d by Dr. Vorpagel i n another c h a p t e r i n t h i s volume. The b e s t d e v e l o p e d example o f t h i s p r o c e s s w i t h p e s t i c i d e s c u r r e n t l y l i e s i n the h e r b i c i d e a r e a . T r i a z i n e s , u r e a s , and many o t h e r h e r b i c i d e s i n h i b i t p h o t o s y n t h e s i s by c o m p e t i t i v e l y d i s p l a c i n g p l a s t o q u i n o n e s from t h e i r b i n d i n g s i t e i n photosystem I I , t h u s d i v e r t i n g e l e c t r o n f l o w from i t s normal pathway and r e s u l t i n g i n the g e n e r a t i o n o f r e a c t i v e i n t e r m e d i a t e s t h a t are c y t o t o x i c (.19,52). R e c e n t l y the p o s i t i o n o f t h i s b i n d i n g s i t e has been l o c a l i z e d t o a 32 kDa p o l y p e p t i d e and t h e m o l e c u l a r a r c h i t e c t u r e o f the s i t e has been e l u c i d a t e d u s i n g such t e c h n i q u e s a s p h o t o a f f i n i t y l a b e l l i n g , gene s e q u e n c i n g o f h e r b i c i d e - r e s i s t a n t m u t a n t s , the development o f a p l a u s i b l e model o f the amino a c i d sequence and i t s f o l d i n g w i t h i n the membrane, and comparison w i t h the s t r u c t u r e o f the r e l a t e d r e g i o n i n the b a c t e r i a l photosystem determined by X-ray c r y s t a l l o g r a p h y



F i g u r e 3. R e l a t i v e p o t e n c i e s of N - p h t h a l y l - L - v a l i n e a n i l i d e , L - v a l i n e , the i m i d a z o l i n o n e h e r b i c i d e , S c e p t e r , and the s u l f o n y l u r e a h e r b i c i d e , c h l o r s u l f u r o n , as i n h i b i t o r s o f a c e t o h y d r o x y a c i d synthase from Zea mays. (Reproduced w i t h p e r m i s s i o n from Ref. 53. C o p y r i g h t 1985 V e r l a g der Z e i t s c h r i f t f u r N a t u r f o r s c h u n g . )

-J

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(52,56,57; F i g u r e 4 ) . T h i s i n f o r m a t i o n i s now b e i n g used i n i n d u s t r y t o design novel i n h i b i t o r s f o r t h i s s i t e as p o t e n t i a l h e r b i c i d e s . R e t u r n i n g t o the t o p i c o f ACh r e c e p t o r s , p r e l i m i n a r y t h r e e d i m e n s i o n a l models are a v a i l a b l e f o r t h i s r e c e p t o r from Torpedo e l e c t r o p l a x ( 5 8 , 5 9 ) . F i g u r e 5 shows the p o s s i b l e s t r u c t u r e o f the ACh b i n d i n g s i t e and t h e r e s i d u e s l i k e l y t o be i n v o l v e d i n ACh b i n d i n g . C o n s i d e r a b l e p r o g r e s s can be expected i n d e v e l o p i n g the s t r u c t u r e o f t h e comparable r e c e p t o r from t h e l o c u s t CNS ( 6 0 ) . N o t a b l e d i f f e r e n c e s e x i s t i n the s u b u n i t s t r u c t u r e o f t h e s e two r e c e p t o r s , a l t h o u g h t h e y may not extend t o a l l t y p e s o f mammalian ACh r e c e p t o r s . I n f o r m a t i o n o f t h i s t y p e may e v e n t u a l l y a l l o w the d e s i g n o f a g o n i s t s and a n t a g o n i s t s h a v i n g a h i g h degree o f s p e c i f i c i t y f o r i n s e c t r e c e p t o r s . In a d d i t i o n t o r e v e a l i n g the topography o f the ACh b i n d i n g s i t e , i t i s p o s s i b l e t h a t such s t u d i e s w i l l r e v e a l new b i n d i n g a r e a s a t which l i g a n d s c o u l d e i t h e r t r i g g e r or i n h i b i t the normal r e c e p t o r - a c t i v a t e d opening o f i o n c h a n n e l . The i o n c h a n n e l i t s e l f o f f e r s such an o p p o r t u n i t y s i n c e s e v e r a l p h a r m a c o l o g i c a l l y a c t i v e agents have been shown t o i n t e r a c t w i t h t h i s pore (58) i n c l u d i n g some i n s e c t i c i d e s ( 6 1 ) . In the a r e a o f f u n g i c i d e s , the b i n d i n g s i t e f o r b e n z i m i d a z o l e f u n g i c i d e s on f u n g a l b e t a - t u b u l i n i n b o t h s e n s i t i v e and r e s i s t a n t f u n g i i s now b e g i n n i n g t o be understood a t t h e m o l e c u l a r l e v e l ( 2 8 ) . A d d i t i o n a l l y , a group o f enzymes w i t h g r e a t s i g n i f i c a n c e f o r p e s t i c i d e a c t i o n and r e s i s t a n c e i s t h e cytochrome P-450 based f a m i l y o f monooxygenases. These a c t b o t h a s a major m e t a b o l i c f o r c e f o r p e s t i c i d e s , and a s a p o t e n t i a l t a r g e t s i t e e.g. t h e y are the e s t a b l i s h e d s i t e o f a c t i o n f o r many f u n g i c i d a l e r g o s t e r o l b i o s y n t h e s i s i n h i b i t o r s and a p o t e n t i a l s i t e f o r i n h i b i t o r s o f j u v e n i l e hormone b i o s y n t h e s i s i n i n s e c t s ( 1 9 ) . I t i s t h e r e f o r e s i g n i f i c a n t t h a t the s t r u c t u r e o f one form o f P-450 has r e c e n t l y been r e v e a l e d by X-ray c r y s t a l l o g r a p h y ( 6 2 ) . F u r t h e r developments a l o n g t h e s e l i n e s c o u l d e v e n t u a l l y open the way f o r the c o m p u t e r - a s s i s t e d d e s i g n o f s e v e r a l t y p e s o f p e s t i c i d e s , growth r e g u l a t o r s and p e s t i c i d e s y n e r g i s t s . A p p l i c a t i o n s o f t h i s approach t o a i d i n d e v e l o p i n g i n h i b i t o r s o f s t e r o l b i o s y n t h e s i s i n f u n g i (63) and g i b b e r e l l i n b i o s y n t h e s i s i n p l a n t s (64) have been d e s c r i b e d . The i n f o r m a t i o n r e g a r d i n g t h e s e and o t h e r b i n d i n g s i t e s s h o u l d be i n v a l u a b l e f o r t h e d e s i g n o f improved and new t y p e s o f l i g a n d s . At p r e s e n t the d i s c o v e r y and o p t i m i z a t i o n o f e f f e c t o r s i s v e r y much a h i t and m i s s phenomenon, a k i n t o t h e o l d game o f p i n n i n g the t a i l on a donkey w h i l e b l i n d f o l d e d . To have the 3 - d i m e n s i o n a l s t r u c t u r e and r e a c t i o n mechanism o f a c r i t i c a l s i t e f u l l y e l u c i d a t e d i s t o remove the b l i n d f o l d . To a p p r e c i a t e the p o t e n t i a l o f t h i s s i t e - d i r e c t e d m o l e c u l a r d e s i g n one has o n l y t o remember t h a t , by adding t o an e x i s t i n g m o l e c u l e a s t r a t e g i c a l l y l o c a t e d f u n c t i o n a l group which forms an a d d i t i o n a l hydrogen bond w i t h t h e r e c e p t o r s u r f a c e , one can i n c r e a s e i t s a f f i n i t y by two o r t h r e e o r d e r s o f magnitude ( 6 5 ) . U n f o r t u n a t e l y , knowledge o f most p o t e n t i a l t a r g e t s i t e s a t t h i s s o p h i s t i c a t e d l e v e l i n most t a r g e t s p e c i e s i s v e r y l i m i t e d o r c o m p l e t e l y l a c k i n g and a g r e a t d e a l o f e f f o r t w i l l be needed t o develop i t t o a usable l e v e l . New Concepts i n Pest C o n t r o l - The D i s c o v e r y o f Pest V u l n e r a b i l i t y . F u r t h e r back i n the c h a i n o f d i s c o v e r y , but o f extreme i m p o r t a n c e f o r f u t u r e developments, s t u d i e s o f p e s t b i o l o g y , p h y s i o l o g y and


6.

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Ser 64

?

I

2

— N H — CH — CO — NH—-CH — CO — NH— CH — CO

-CO—

—

CH— N H - C O — C H — N H -

k

I

Figure 4. Proposed plastoquinine (QB) and herbicide binding s i t e on the 32 kDalton D-1 polypeptide of photosystem I I . The quinone i s bound through an iron-complexed h i s t i d i n e residue (his 215) and hydrogen bonding to ser 264. Further interactions occur with arg 269 and phe 255 lying above and below the binding s i t e . Amino acid substitutions in herbicide-tolerant mutants have been i d e n t i f i e d at the residues numbered 219, 255, 264 and 275. Reproduced with permission from Ref. 57. Copyright 1986 Verlag der Z e i t s c h r i f t fur Naturforschung.


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Threonine-133

Aspartate-138

Glutamine-140

lsoleucine-131

129

1

Cysteine-128

Cysteine-142

Figure 5. Model of the proposed acetylcholine recognition s i t e on the alpha subunit of the Torpedo ACh receptor i n the region between Cys 128 and Cys 142. An ACh molecule i s shown i n r e l a t i o n to the four residues postulated to interact i n i t s binding. Reprinted with permission from Ref. 59. Copyright 1985 Elsevier Science Publishers.


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b i o c h e m i s t r y l e a d t o new c o n c e p t s i n p e s t c o n t r o l by d e f i n i n g b a s i c d i f f e r e n c e s between t a r g e t and n o n - t a r g e t s p e c i e s . As examples, the d i s c o v e r y o f the c o n t r o l o f i n s e c t development by j u v e n i l e hormones, and o f mating and a g g r e g a t i o n b e h a v i o r s by i n s e c t pheromones, were e a g e r l y and r a p i d l y e x p l o i t e d f o r new and v e r y s a f e approaches t o i n s e c t c o n t r o l . These a r e a s c o n t i n u e t o a t t r a c t r e s e a r c h aimed a t n o v e l a p p l i c a t i o n s o f t h i s b a s i c knowledge. Even r e s t r i c t i n g the d i s c u s s i o n t o i n s e c t p e s t s , many o t h e r s i g n i f i c a n t d i f f e r e n c e s i n t h e i r b i o c h e m i s t r y and p h y s i o l o g y compared t o v e r t e b r a t e s are a l r e a d y known. These r e p r e s e n t p o t e n t i a l a r e a s f o r the development o f s a f e r p e s t i c i d e s , some o f which are c u r r e n t l y under study w i t h t h i s end i n v i e w e.g. p e c u l i a r i t i e s o f s t e r o l m e t a b o l i s m i n i n s e c t s , t h e i r r e l i a n c e on octopamine and s p e c i f i c n e u r o p e p t i d e s as n e u r o e f f e c t o r s , and t h e i r c u t i c u l a r b i o c h e m i s t r y and w a t e r p r o o f i n g (45,46,48,66). F u r t h e r , s e l e c t i v i t y and mammalian s a f e t y can be a c h i e v e d j u s t as r e a d i l y by t h e development o f compounds which are degraded a t d i f f e r e n t i a l r a t e s i n f r i e n d and foe as by a t t a c k i n g b i o c h e m i c a l t a r g e t s p e c u l i a r t o the p e s t , as i l l u s t r a t e d by the p y r e t h r o i d i n s e c t i c i d e s . Lack o f a b i l i t y t o r a p i d l y degrade a t o x i c a n t i s c l e a r l y a s e r i o u s v u l n e r a b i l i t y f a c t o r . There i s a g r e a t need f o r s t u d i e s on the b a s i c p h y s i o l o g y and b i o c h e m i s t r y o f a l l t y p e s o f p e s t s t o c o n t i n u e and be expanded i n o r d e r t o b e t t e r understand e x i s t i n g s i t e s o f v u l n e r a b i l i t y and t o d i s c o v e r new ones which can lead to novel concepts f o r c o n t r o l . To p r o v i d e but a s i n g l e example from many, the d e f e n s e r e s p o n s e s o f i n s e c t s a g a i n s t i n v a d i n g pathogens r e p r e s e n t s j u s t such a p r o m i s i n g t o p i c o f c u r r e n t r e s e a r c h . R e s u l t s i n t h i s a r e a have r e c e n t l y been reviewed by Dunn ( 6 7 ) . In l e p i d o p t e r o u s l a r v a e , t h e s e d e f e n s e s are m u l t i p l e and i n c l u d e b o t h humoral and c e l l u l a r elements. Invading organisms are s u b j e c t t o p h a g o c y t o s i s and e n c a p s u l a t i o n i n c e l l u l a r networks d e r i v e d from hemocytes. Over a p e r i o d o f 24 t o 48 h o u r s , these i n i t i a l d e f e n s e s are augmented by the s y n t h e s i s and r e l e a s e i n t o t h e blood o f lysozyme and l y s i n e - and a r g i n i n e - r i c h p o l y p e p t i d e a n t i b i o t i c s ( b a c t e r i c i d i n s ) from the f a t body. I t i s premature t o suggest t h a t such i n f o r m a t i o n c u r r e n t l y p r o v i d e s o b v i o u s o p p o r t u n i t i e s f o r e x p l o i t a t i o n i n i n s e c t c o n t r o l , but the p o t e n t i a l i s c l e a r . I f means c o u l d be found t o t u r n o f f or o b v i a t e some or a l l o f t h e s e d e f e n s e s , the i n s e c t would be more l i k e l y t o succumb t o n a t u r a l i n f e c t i o n s . T h i s c o u l d be p a r t i c u l a r l y u s e f u l i f a p p l i e d i n c o m b i n a t i o n w i t h an i n f e c t i o u s agent such as a b a c t e r i u m or v i r u s . Such a concept i s more than a pipe-dream s i n c e i t has been shown t h a t a v i r u s a s s o c i a t e d w i t h the eggs o f p a r a s i t i c wasps p r e v e n t s the e n c a p s u l a t i o n response o f the host which would o t h e r w i s e tend t o o c c l u d e the egg (68.). An u n i d e n t i f i e d component from the venom o f o t h e r p a r a s i t i c wasps has a l s o shown immunosuppressant p r o p e r t i e s i n i n s e c t s (6_9) and two immunosuppressants a c t i v e on g i a n t s i l k w o r m moths have been d e t e c t e d i n p r e p a r a t i o n s o f B a c i l l u s t h u r i n g i e n s i s ( 7 0 ) . F u r t h e r , Dunn and h i s coworkers have found t h a t q u i t e s m a l l p e p t i d o g l y c a n fragments o f the b a c t e r i a l c e l l w a l l are r e c o g n i z e d as " f o r e i g n " i n Manduca s e x t a and w i l l t r i g g e r the p r o d u c t i o n o f humoral d e f e n s e s . Can we f i n d low m o l e c u l a r weight a n a l o g s t o b l o c k t h e s e " r e c e p t o r " s i t e s and thus i n h i b i t t h i s a s p e c t o f immunity? F i n a l l y , t h e d e f e n s i v e a n t i b i o t i c s are r i c h i n a r g i n i n e . Canavanine, an analog o f a r g i n i n e which o c c u r s i n some legume seeds, can be t o x i c by r e p l a c i n g a r g i n i n e i n e s s e n t i a l p r o t e i n s and p e p t i d e s ( 7 1 ) .



72

H N-C-NHOCH CH CH(NH2)COOH 2

2

2

Canavanine

Manduca l a r v a e t r e a t e d w i t h c a n a v a n i n e produce a n t i b i o t i c p e p t i d e s i n which a r g i n i n e i s r e p l a c e d by c a n a v a n i n e . T h e i r i n t r i n s i c b i o l o g i c a l a c t i v i t y i s g r e a t l y d i m i n i s h e d and t h e s e l a r v a e may be u n u s u a l l y s u s c e p t i b l e t o i n f e c t i o n by b a c t e r i a (P. E. Dunn, p e r s o n a l communication). I t i s o n l y r e a l i s t i c t o admit t h a t t h e chances o f e v e n t u a l l y b e i n g a b l e t o e x p l o i t any such example o f b a s i c r e s e a r c h a r e h i g h l y u n c e r t a i n and r e q u i r e a l o n g l e a d time f o r development. Even t h e d i s c o v e r y o f mechanisms t o d i s r u p t e s s e n t i a l b i o c h e m i c a l p r o c e s s e s p e c u l i a r t o pest groups does n o t o f f e r a panacea f o r s e v e r a l r e a s o n s . 1. Though we may a v o i d acute t o x i c i t y t o most n o n - t a r g e t s p e c i e s , t h e r e a r e no g u a r a n t e e s t h a t we w i l l a v o i d c h r o n i c t o x i c i t y : t h e two are n o t c o r r e l a t e d . In f a c t t h e r e may be a n e g a t i v e c o r r e l a t i o n s i n c e c u r r e n t p r o t o c o l s f o r s a f e t y e v a l u a t i o n employ t h e maximum t o l e r a t e d dose i n l o n g term s t u d i e s w i t h v e r t e b r a t e s . Hence compounds which c a n be g i v e n a t h i g h d o s e s because o f t h e i r low a c u t e t o x i c i t y a r e a t a disadvantage i n l i f e t i m e chronic t e s t s . 2. Though t h e s i t e o f a c t i o n may be p e c u l i a r t o t h e p e s t , t h e r e may be p a r a l l e l p r o c e s s e s i n n o n - t a r g e t s p e c i e s t h a t a l s o a r e affected. 3. Even i f a f t e r enormous e f f o r t and i n v e s t m e n t a new approach c l e a r s a l l t h e s e h u r d l e s and c a n be used i n p r a c t i c e , we s h a l l f i n d no v i c t o r y over p e s t s t o be permanent. The l o s s o f v u l n e r a b i l i t y t h r o u g h t h e development o f r e s i s t a n c e w i l l always be a t h r e a t e n i n g p o s s i b i l i t y . T h i s i s why i t i s so c r u c i a l t h a t much g r e a t e r e f f o r t be devoted t o u n d e r s t a n d i n g t h e p o p u l a t i o n , g e n e t i c , and b i o c h e m i c a l b a s i s o f t h e development o f r e s i s t a n c e t o c o n t r o l measures i n general. However, on t h e b a s i s o f p a s t e x p e r i e n c e , i t i s r e a s o n a b l e t o c o n c l u d e t h a t w i t h f a i t h , p a t i e n c e , and a s u f f i c i e n t i n v e s t m e n t , a t l e a s t some such b a s i c s t u d i e s o f p e s t b i o c h e m i s t r y w i l l pay o f f i n n o v e l and s a f e r p e s t c o n t r o l s t r a t e g i e s . The

Clouded

Future

S i n c e t h e Second World War t h e US h a s i n v e s t e d l a r g e amounts o f money on r e s e a r c h i n t o b a s i c b i o l o g i c a l mechanisms and t h e i r c o n t r o l , l a r g e l y s p u r r e d by m e d i c a l g o a l s , b u t a l s o , t o a l e s s e r e x t e n t , by t h e m o t i v a t i o n t o understand p o t e n t i a l p e s t s and t h e i r e s s e n t i a l c h a r a c t e r i s t i c s . T h i s i n v e s t m e n t by government and i n d u s t r y h a s proved r e m a r k a b l y f r u i t f u l . There i s r e a s o n t o hope t h a t we a r e now on t h e t h r e s h o l d o f an e r a where we w i l l be a b l e t o d e s i g n compounds t h a t a r e b o t h h i g h l y p o t e n t and h i g h l y s p e c i f i c i n t h e i r t o x i c e f f e c t s , and t o p r e v e n t r e s i s t a n c e from n e g a t i n g them. A l t h o u g h much remains t o be done t o a c h i e v e t h e s e ends t h e r e i s cause f o r optimism t h a t we can a c h i e v e them. However, such advances, s t a r t i n g from b a s i c s t u d i e s , a r e i n c r e a s i n g l y c o s t l y and demand a team e f f o r t


6.

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and c o o p e r a t i o n between i n d u s t r y and academia. T h i s comes a t a time when t h e agrochemical i n d u s t r y i s e x p e r i e n c i n g lowered p r o f i t s and a decreased a b i l i t y t o i n v e s t i n such b a s i c and e x p e n s i v e r e s e a r c h . A t h i r d e s s e n t i a l p a r t n e r i n t h i s p r o c e s s i s government, p a r t i c u l a r l y , because o f i t s a g r i c u l t u r a l mandate, t h e USDA. However, governmental r e s e a r c h support i s a l s o e x p e r i e n c i n g f i n a n c i a l s t r i n g e n c y and uncertainty. The USDA, even i n the p a s t , has n o t , from the p e r s p e c t i v e o f t h i s a r t i c l e , i n v e s t e d i t s l i m i t e d r e s o u r c e s o p t i m a l l y . When i t became c l e a r i n the 1960 s t h a t s o c i e t y c o r r e c t l y regarded many c u r r e n t p e s t i c i d e s as too flawed f o r c o n t i n u e d use, two l o g i c a l approaches t o the development o f s a f e r p e s t c o n t r o l s t r a t e g i e s c o u l d be e n v i s i o n e d : (1) t o d e v e l o p a l t e r n a t i v e s t o c h e m i c a l p e s t i c i d e s , and (2) t o support b a s i c r e s e a r c h aimed towards the d i s c o v e r y o f new and s a f e r c h e m i c a l compounds t o r e p l a c e those t h a t were u n s a t i s f a c t o r y . The USDA chose t o emphasize the former approach h e a v i l y a t t h e expense o f t h e l a t t e r . Though p o l i t i c a l l y p o p u l a r , t h i s may have been s h o r t - s i g h t e d , and has not r a d i c a l l y changed our approach t o p e s t c o n t r o l which c o n t i n u e s t o depend v e r y h e a v i l y on p e s t i c i d e s . Meanwhile f u n d i n g from t h i s p o t e n t i a l l y p r i m a r y s o u r c e t o support research i n t o the b e t t e r understanding o f the biochemical s i t e s and modes o f a c t i o n o f p e s t i c i d e s , t o a i d i n the d i s c o v e r y o f new methods t o d e v e l o p improved and s a f e r compounds, and t o spur e f f o r t s t o understand and a l l e v i a t e r e s i s t a n c e t o p e s t i c i d e s has been n e g l i g i b l e i n comparison t o the needs and o p p o r t u n i t i e s . T h i s v i e w p o i n t has been i n d e p e n d e n t l y expressed by o t h e r s a l s o ( 4 8 ) . I t i s to be hoped t h a t the emphasis on s t r a t e g i e s f o r d e v e l o p i n g s a f e r pest c o n t r o l t e c h n o l o g i e s can soon be brought i n t o a more r a t i o n a l balance. When one c o n s i d e r s the immense sums b e i n g i n v e s t e d i n weapons and m e d i c a l r e s e a r c h , o r elsewhere i n a g r i c u l t u r e , the amount needed t o support such work a t a r e a l i s t i c l e v e l i s m i n i s c u l e . J u s t 0.01% o f t h e amount expected t o be spent t o s u b s i d i z e a g r i c u l t u r a l p r o d u c t i o n i n the USA i n 1986 would p r o v i d e $3 m i l l i o n as a f i r m f o u n d a t i o n f o r p e s t i c i d e - r e l a t e d b a s i c r e s e a r c h . T h i s same amount r e p r e s e n t s 2% o f what i s spent t o m a i n t a i n m i l i t a r y bands i n the USA. C u r r e n t l y , t h e r e i s a minimum o f encouragement o r o p p o r t u n i t y i n the USA t o work on t h e s e t o p i c s , and a l a r g e p r o p o r t i o n o f t h e r e s e a r c h i s done i n o t h e r n a t i o n s . At the r e c e n t IUPAC I n t e r n a t i o n a l Congress o f P e s t i c i d e C h e m i s t r y , w h i c h a t t r a c t e d a worldwide attendance and was h e l d c l o s e to the USA i n Canada, 78% o f the papers on f u n g i c i d e modes o f a c t i o n were g i v e n by speakers w i t h non-US a f f i l i a t i o n s , a s were 89% o f the papers on h e r b i c i d e modes o f a c t i o n , and 82% o f t h o s e on f u n g i c i d e and i n s e c t i c i d e r e s i s t a n c e . W h i l e i t i s encouraging t h a t such work i s thought worthy o f support i n many n a t i o n s around the w o r l d , t h e c l e a r i n d i c a t i o n i s t h a t , d e s p i t e i t s huge a g r i c u l t u r a l i n d u s t r y w i t h a c o n t i n u i n g dependence on p e s t i c i d e s , the USA i s l a g g i n g s e r i o u s l y i n such n e c e s s a r y r e s e a r c h e f f o r t s . The c o m b i n a t i o n o f f i n a n c i a l s t r e s s e s t h a t c u r r e n t l y t h r e a t e n a l l s o u r c e s o f f u n d i n g f o r a g r i c u l t u r a l r e s e a r c h i s i n danger o f l e a v i n g us w i t h f r u i t r i p e f o r the p i c k i n g as a r e s u l t o f our p r i o r i n v e s t m e n t s i n b i o l o g i c a l r e s e a r c h , but w i t h few h a r v e s t e r s i n t h e o r c h a r d . I f s o , o t h e r s may then p r o f i t from our l a c k o f v i s i o n . f



74 Acknowledgments

My thanks go to Drs. P. E. Dunn and J . V. Osmun for c r i t i c a l l y reading t h i s manuscript, and to Dr. M. E. Schroeder for providing unpublished information regarding the discovery of the nitromethylene heterocyclic i n s e c t i c i d e s . Journal Paper No. 10994, A g r i c u l t u r a l Experiment Station, Purdue University, W. Lafayette, IN 47907.

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19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43.

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