Insecticides of Plant Origin - American Chemical Society

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on June 5, 2015 | http://pubs.acs.org Publication Date: February 23, 1989 | doi: 10.1021/bk-1989-0387.ch005

Chapter 5

Nonprotein Amino Acid Feeding Deterrents from Calliandra 1

2

John T. Romeo and Monique S. J . Simmonds 1

Department of Biology, University of South Florida, Tampa, F L 33620 Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3AB, England 2

Several nonprotein imino acids and sulphur amino acids are found in seeds, leaves and sap of Calliandra, a tropical Mimosoid legume. Concentrations may reach 3% dry weight. Individual imino acids exhibit modest insecticidal activity against some lepidopterans, affecting all stages of the life cycle. Varying degrees of mortality are seen in Spodoptera and Heliothis species, and delayed growth and decreased fecundity are also common. S-(β-carboxyethyl)-cysteine and cis-4hydroxypipecolic acid are deterrent to feeding by these insects. In aphids, combinations of imino and sulphur amino acids, which mimic those found in the plants, are deterrent to feeding, and they also reduce survival and fecundity. Electrophysiological, behavioral, and nutritional studies have not yet clarified the mode of action of these compounds. The t r o p i c a l Mimosoid legume genus C a l l i a n d r a c o n s i s t s o f some 200 s p e c i e s c o n f i n e d m o s t l y t o t h e New World. The p l a n t s range from woody shrubs t o medium s i z e t r e e s , and have n o t y e t been e x p l o i t e d on a l a r g e s c a l e . I n I n d o n e s i a , however, the r a p i d l y growing C. calothyrsus i s widely c u l t i v a t e d f o r firewood. I t i s recommended as a supplementary p l a n t i n v i l l a g e s and r u r a l a r e a s f o r use as f u e l and f o d d e r so t h a t n a t u r a l f o r e s t s c a n be s p a r e d d e s t r u c t i o n . F o l i a g e c o n t a i n s up t o 22 p e r c e n t p r o t e i n and c a t t l e and g o a t s consume i t f r e e l y . Bees u s e i t s n e c t a r f o r p r o d u c i n g honey ( 1 ) . W h i l e as y e t u n t e s t e d , o t h e r s p e c i e s a r e l i k e l y t o p r o v e u s e f u l i n the humid t r o p i c s , and C a l l i a n d r a has been d e s i g n a t e d as one o f e i g h t p o t e n t i a l l y e x p l o i t a b l e t r o p i c a l p l a n t groups n e e d i n g f u r t h e r scientific investigation(2). 0097-6156/89/0387-0059$06.00/0 © 1989 American Chemical Society

In Insecticides of Plant Origin; Arnason, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

INSECTICIDES OF PLANT ORIGIN

60


N o n p r o t e i n Amino A c i d C h e m i s t r y o f C a l l i a n d r a The f o l i a g e of C a l l i a n d r a i s known to c o n t a i n an a r r a y o f r a r e n o n p r o t e i n i m i n o a c i d s (amino a c i d s c o n t a i n i n g a h e t e r o c y c l i c n i t r o g e n r i n g ) which are l a r g e l y c o n f i n e d t o legumes. The compounds are d e r i v a t i v e s o f p i p e c o l i c a c i d , the h i g h e r homologue o f p r o l i n e , and i n c l u d e an a c e t y l a m i n o , f o u r monohydroxy, and f o u r d i h y d r o x y d e r i v a t i v e s (Figure 1). D i s t r i b u t i o n p a t t e r n s a r e s p e c i e s s p e c i f i c and u s u a l l y the monohydroxy p r e c u r s o r s a r e found t o g e t h e r w i t h one or two d i h y d r o x y compounds ( 3 ) . In C a l l i a n d r a , p i p e c o l i c a c i d i s s y n t h e s i z e d from l y s i n e and the imino r i n g i s s u b s e q u e n t l y h y d r o x y l a t e d t o form the mono and d i h y d r o x y d e r i v a t i v e s ( 4 ) . There i s some m e t a b o l i c i n t e r c o n v e r s i o n o f imino compounds w i t h i n a g i v e n p l a n t , but i n d i v i d u a l c o n c e n t r a t i o n s remain r e m a r k a b l y s t a b l e and u s u a l l y range between 0.1-0.5% d r y weight of the p l a n t . The compounds o c c u r i n s i m i l a r c o n c e n t r a t i o n i n seeds. Seeds a l s o c o n t a i n a group o f r a r e n o n p r o t e i n s u l p h u r amino a c i d s d e r i v e d from c y s t e i n e . The major s u l p h u r compound i s S-(B -carboxyethyl)-cysteine (S-CEC) which o f t e n a c c o u n t s f o r up to 3% of the t o t a l p l a n t d r y weight ( F i g u r e 1 ) . L e s s e r amounts of S-(3 - c a r b o x y i s o p r o p y l ) - c y s t e i n e , d j e n k o l i c a c i d and N - a c e t y l d j e n k o l i c a c i d are a l s o present. The s u l p h u r amino a c i d s a r e m e t a b o l i z e d upon g e r m i n a t i o n but p e r s i s t i n newly emerged l e a v e s and sap f o r t e n weeks o r more ( 5 ) . In Z a p c t e c a , a r e l a t e d genus endemic to Mexico and f o r m e r l y p a r t of C a l l i a n d r a , the s u l p h u r amino a c i d s are m e t a b o l i z e d t o v o l a t i l e compounds which a r e exuded from the r o o t s . Objectives

of

Research

Because C a l l i a n d r a i s a t r o p i c a l legume w i t h p o t e n t i a l f o r economic e x p l o i t a t i o n , because i t i s u n u s u a l i n s y n t h e s i z i n g two d i f f e r e n t groups o f r a r e n o n p r o t e i n amino a c i d s , and b e c a u s e , r e l a t i v e t o a s s o c i a t e d p l a n t s , many s p e c i e s s u f f e r m i n i m a l i n s e c t prédation, i t has been the f o c u s o f r e s e a r c h i n the l a b o r a t o r y o f the s e n i o r author f o r s e v e r a l years. U s i n g a v a r i e t y o f t e c h n i q u e s and b i o a s s a y s , e x p e r i m e n t s have been conducted t o a s s e s s what e c o l o g i c a l s i g n i f i c a n c e the amino a c i d s have as f e e d i n g d e t e r r e n t s and p o t e n t i a l i n s e c t i c i d a l agents. Feeding Experiments - Lepidopterans C a l l i a n d r a l e a f powder i n c o r p o r a t e d i n t o a r t i f i c i a l p i n t o bean based agar d i e t s a t 2.5, 5, and 10% l e v e l s and f e d t o S p o d o p t e r a f r u g i p e r d a ( f a l l armyworm) produces a v a r i e t y o f e f f e c t s on a l l p a r t s o f the i n s e c t l i f e c y c l e ( 6 ) . S i g n i f i c a n t m o r t a l i t y occurs i n the l a r v a l s t a g e . Of t h e s e i n s e c t s which s u r v i v e , the l a r v a l w e i g h t s are reduced and t h e r e i s a d e l a y i n p u p a t i o n o f s e v e r a l days. P u p a l and emerging a d u l t w e i g h t s a r e a l s o reduced as i s the p e r c e n t a g e of a d u l t moths emerging. There are no apparent malformed l a r v a e or pupae. To some e x t e n t o l d e r l a r v a e are a b l e t o make up e a r l y weight l o s s . The n o n p r o t e i n amino a c i d s o f C a l l i a n d r a a r e m o d e r a t e l y t o x i c i n s i m i l a r f e e d i n g t e s t s . When an aqueous e x t r a c t o f l e a v e s , from which a l l compounds o t h e r t h a n the amino a c i d s have been removed, i s



R O M E O & SIMMONDS

Pipecolic acid;

Feeding Deterrents from Calliandra

=

=

=

=H

Çis-4-Hydroxypipecolic a c i d ; R^ = OH, R^ = R^ = R^ = H Trans-4-Hydroxypipecolic a c i d ; R = OH,

= R = R^, « H

2

3

Çis-5-Hydroxypipecolic a c i d ; R = OH, R^ = R^ = R^ = H 3

Trans-5-Hydroxypipecolic a c i d ; R^ = OH, R^ = R = R^ = H 2

Trans-Trans-4,5-Dihydroxypipecolic a c i d ; R • R = OH, I 2

Trans-Cis-4,5-Dihydroxypipecolic a c i d ;

3

-

- OH, Rj

C i s - C i s - 4 , 5 - D i h y d r o x y p i p e c o l i c a c i d ; R = R = OH, R - 1 1

3

2

Cis-Trans-4,5-Dihydroxypipecolic a c i d ; R^ = R^ = OH, R

2

Acetylaminopipecolic a c i d ; R - NHCOCH^, R^ = R^ = R^ = H 2

S-(β-Carboxyethyl)-cysteine

HOOC-CH -CH -S-CH -j:H-COOH 2

2

2

NH

Figure 1.

2

P i p e c o l i c Acid

Derivatives



62


i n c o r p o r a t e d i n t o agar based d i e t a t 100% p l a n t e q u i v a l e n c y , t h e growth o f Spodoptera f r u g i p e r d a i s reduced by 20% ( 7 ) . I n c o r p o r a t i n g i n d i v i d u a l amino a c i d s i n t o the d i e t produces e f f e c t s s i m i l a r t o those o f e x t r a c t s from ground l e a v e s , however, the r e s u l t s a r e v a r i a b l e and one cannot p r e d i c t a p r i o r i which compound i s l i k e l y t o be most t o x i c ( T a b l e I ) . A t l e v e l s a t which they e x i s t i n p l a n t s (0.1-0.5%) the r e l a t i v e l y common t r a n s - 5 - h y d r o x y p i p e c o l i c a c i d i s more t o x i c t o S^ f r u g i p e r d a t h a n e i t h e r the r a r e t r a n s - t r a n s - 4 , 5 , d i h y d r o x y p i p e c o l i c a c i d or a c e t y l a m i n o p i p e c o l i c a c i d . The l a t t e r compounds e x h i b i t t o x i c i t y o n l y a t h i g h e r l e v e l s o f 1 t o 2.5%. The t o x i c i t y o f t r a n s - c i s - 4 , 5 d i h y d r o x y p i p e c o l i c a c i d i s e x p r e s s e d a t 0.5%, t h e upper l e v e l o f i t s c o n c e n t r a t i o n i n p l a n t s . In o t h e r experiments u s i n g f i r s t stadium l a r v a e o f Spodoptera l i t t o r a l i s , H e l i o t h i s v i r e s c e n s and H e l i o t h i s a r m i g e r a , i n d i v i d u a l imino compounds and S-CEC were i n c o r p o r a t e d a t 0.5% i n t o bean based d i e t s t o l o o k f o r m o r t a l i t y e f f e c t s ( T a b l e I I ) . L a r v a e o f S. littoralis were more s u s c e p t i b l e t o imino a c i d s t h a n the l a r v a e o f Heliothis. Three imino a c i d s and the s u l p h u r amino a c i d were effective. S-CEC was the o n l y amino a c i d t o cause s i g n i f i c a n t m o r t a l i t y to armigera. The e f f e c t o f imino a c i d s on the f e e d i n g b e h a v i o r o f f i n a l stadium l a r v a e o f l i t t o r a l i s , H. v i r e s c e n s , and a r m i g e r a was a s s e s s e d by p r e s e n t i n g the compounds i n c o m b i n a t i o n w i t h the p h a g o s t i m u l a n t s u c r o s e on g l a s s f i b e r d i s c s . L a r v a e , 24-48 hours i n t o the f i n a l s t a d i u m , were d e p r i v e d o f food f o r 4 hours and p l a c e d i n d i v i d u a l l y i n P e t r i d i s h e s c o n t a i n i n g a c o n t r o l and treatment disc. Each d i s c c o n t a i n e d s u c r o s e a t a c o n c e n t r a t i o n o f 0.01% w/w. The t r e a t m e n t d i s c s were t r e a t e d a d d i t i o n a l l y w i t h t h e t e s t s o l u t i o n at 100 ppm. D i s c s were weighed a t the b e g i n n i n g and t e r m i n a t i o n o f the e x p e r i m e n t . The b i o a s s a y was t e r m i n a t e d when 50% o f any one d i s c was e a t e n ( u s u a l l y a f t e r 8-12 h o u r s ) . An A n t i f e e d a n t Index f ( C - T ) / ( C + T ) % ] , where C i s weight o f c o n t r o l and Τ o f treatment d i s c , was c a l c u l a t e d on the amounts e a t e n . V a l u e s i n t h i s Index range from +100, a p o t e n t a n t i f e e d a n t , t o -100 a p h a g o s t i m u l a n t . T a b l e I I I shows t h a t o f t h e compounds t e s t e d o n l y c i s - 4 h y d r o x y p i p e c o l i c a c i d (C4) and S-CEC s i g n i f i c a n t l y d e c r e a s e d f e e d i n g , and d i d so i n a l l t h r e e s p e c i e s . The o t h e r compounds were e i t h e r i n a c t i v e ( i . e . CT) o r a c t u a l l y had some p h a g o s t i m u l a n t a c t i v i t y ( i . e . C5 and CC) . The b e h a v i o r o f Sj_ l i t t o r a l i s was s t u d i e d i n more d e t a i l d u r i n g the f i r s t 2 h o u r s of exposure t o the d i s c s by u s i n g a Video camera and measuring the f o l l o w i n g p a r a m e t e r s : time t o c o n t a c t o f t h e treatment d i s c , d u r a t i o n o f t h i s c o n t a c t , and amount o f time spent f e e d i n g on the treatment and c o n t r o l d i s c s . S-CEC was the o n l y compound t o s i g n i f i c a n t l y d e c r e a s e t h e amount o f time spent f e e d i n g during the i n i t i a l p e r i o d of the a n t i f e e d a n t assay. I n s e c t s spent 40% o f t h e time on c o n t r o l d i s c s v s 11% on treatment d i s c s . The a n t i f e e d a n t a c t i v i t y o f t h i s compound was m a i n t a i n e d d u r i n g the r e s t o f t h e a s s a y and t h i s c o u l d have been a f a c t o r c o n t r i b u t i n g t o m o r t a l i t y i n the d e v e l o p m e n t a l experiments ( T a b l e I I ) . I n c o n t r a s t , many o f the imino compounds (C4, C5, T4, and CC) a c t u a l l y s t i m u l a t e d f e e d i n g d u r i n g the f i r s t two h o u r s . I n the case o f C4, however, t h i s s t i m u l a t i o n was l a t e r r e v e r s e d as shown by the A n t i f e e d a n t Index. By the end o f the experiment, s i g n i f i c a n t l y more o f t h e c o n t r o l than t r e a t m e n t d i s c was consumed ( T a b l e I I I ) .


5.



Table I .

Compound T5 TC TC ΑΑΡ TT TT

63


R e l a t i v e T o x i c i t i e s o f N o n p r o t e i n Imino A c i d s To F r e s h l y Hatched Spodoptera f r u g i p e r d a (Adapted from Romeo, 1984) Concentration 0.1% 0.5% 1.0% 1.0% 1.0% 2.5%

Mortality 60%* 30%* 60%* 55%* 10%* 100%*

Growth I n h i b i t i o n Yes Yes Yes Yes Yes

η = 20; T5 = t r a n s - 5 - h y d r o x y p i p e c o l i c a c i d ; TC = t r a n s - c i s 4 , 5 - d i h y d r o x y p i p e c o l i c a c i d ; ΑΑΡ = a c e t y l a m i n o p i p e c o l i c a c i d ; TT = t r a n s - t r a n s - 4 , 5 - d i h y d r o x y p i p e c o l i c a c i d . * S i g n i f i c a n t l y d i f f e r e n t from c o n t r o l

Table I I .

Compound C4 C5 T5 CC TC TT S-CEC

(Tukey's t e s t , ρ < 0.05).

P e r c e n t M o r t a l i t y o f F i r s t Stadium L a r v a e o f Spodoptera and H e l i o t h i s Exposed t o 0.5% C o n c e n t r a t i o n s o f Amino A c i d s S. l i t t o r a l i s 34* 34* 37* 12 15 0 39*

H. v i r e s c e n s 14 10 24 10 12 0 21

H. a r m i g e r a 16 6 21 6 10 0 30*

η = 20; C4 = c i s - 4 - h y d r o x y p i p e c o l i c a c i d ; C5 = c i s - 5 - h y d r o x y p i p e c o l i c a c i d ; T5 = t r a n s - 5 - h y d r o x y p i p e c o l i c a c i d ; CC = c i s c i s - 4 , 5 - d i h y d r o x y p i p e c o l i c a c i d ; TC = t r a n s _ - c i s - 4 , 5 - d i h y d r o x y p i p e c o l i c a c i d ; TT = t r a n s - t r a n s - 4 , 5 - d i h y d r o x y p i p e c o l i c a c i d ; S-CEC = S - ( 3 - c a r b o x y e t h y l ) - c y s t e i n e . * = s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l u n e q u a l sample s i z e s , ρ < 0.05)

(Tukey's t e s t f o r



64


Feeding Experiments

- Aphids

The modest and v a r y i n g e f f e c t s o b s e r v e d thus f a r on b e h a v i o r , growth and m o r t a l i t y do not suggest t h e s e amino a c i d s have p o t e n t i a l as i n s e c t i c i d e s a g a i n s t l e p i d o p t e r a n s . A s t u d y done w i t h a p h i d s (Aphis f a b a e ) , however, p r o v i d e d some i n t e r e s t i n g f i n d i n g s . We used a b i o a s s a y i n w h i c h b o t h i n d i v i d u a l amino a c i d s and c o m b i n a t i o n s were a d m i n i s t e r e d by a l l o w i n g second l e a f s t a g e bean s e e d l i n g s o f P h a s e o l u s v u l g a r i s (from which the l e a v e s and c o t y l e d o n s were removed and the top p o r t i o n s of the stems c u t o f f a t the l e v e l o f the c o t y l e d o n s ) t o absorb them from t e s t s o l u t i o n s . A f t e r 24 hours of e q u i l i b r a t i o n , a p h i d s were c o n f i n e d on t h e s e p l a n t s by p l a c i n g a f i l t e r paper cone around the stem. T h e i r b e h a v i o r was observed f o r two hours and m o r t a l i t y and f e c u n d i t y were measured a f t e r 5 and 10 days. The b i o a s s a y has the advantages t h a t compounds a r e d e l i v e r e d i n a n a t u r a l way and the experiment can be c o n t i n u e d f o r a r e l a t i v e l y long time. A p h i d b e h a v i o r was m o n i t o r e d by c o u n t i n g the number o f p r o b e s , p r o d u c t i o n o f honeydew, movements on the p l a n t , and movements o f f the p l a n t t o the c o n f i n i n g f i l t e r paper cone. Some o f the r e s u l t s a r e shown i n T a b l e s IV & V. No i n d i v i d u a l compound o r 2-way c o m b i n a t i o n s i g n i f i c a n t l y a f f e c t e d f e e d i n g b e h a v i o r . As s o l u t i o n s more c l o s e l y approximated the a c t u a l c o m b i n a t i o n s o f amino a c i d s e n c o u n t e r e d i n C a l l i a n d r a p l a n t s , however, d e t e r r e n c y became a p p a r e n t . Dose dependent c u r v e s were seen c o n s i s t e n t l y when the s u l p h u r amino a c i d (S-CEC) was p r e s e n t t o g e t h e r w i t h one o f the d i h y d r o x y p i p e c o l i c a c i d isomers (Table IV). A f t e r 5 days exposure, c i s - t r a n s - 4 , 5 - d i h y d r o x y p i p e c o l i c a c i d (CT) was the o n l y i n d i v i d u a l amino a c i d t o s i g n i f i c a n t l y d e c r e a s e the number o f a p h i d s on the p l a n t s . However, a l l the t h r e e and f o u r way c o m b i n a t i o n s , which c o n t a i n e d b o t h imino and the s u l p h u r amino a c i d d i d so ( T a b l e V ) . S o l u t i o n s were removed a f t e r f i v e days and r e p l a c e d w i t h water f o r the f i n a l f i v e days. At the end o f the t e n day p e r i o d , the numbers o f a p h i d s were r e c o u n t e d . Toxic e f f e c t s i n t e n s i f i e d during t h i s t i m e , and d e l a y e d e f f e c t s o f some i n d i v i d u a l amino a c i d s became apparent. F o r example, t r a n s - t r a n s - 4 , 5 - d i h y d r o x y p i p e c o l i c a c i d (TT), a compound which had l i t t l e e f f e c t a f t e r 5 days, was s i g n i f i c a n t l y t o x i c a f t e r 10. S i n c e we were a b l e t o d e t e c t the a d m i n i s t e r e d amine a c i d s i n the a p h i d s a t the c o n c l u s i o n o f the experiment, i t would seem we a r e measuring t o x i c r a t h e r than starvation effects. Mode o f A c t i o n A s e a r c h f o r the mechanism o f a c t i v i t y o f t h e s e compounds has p r o v e d elusive. I n g e s t e d n o n p r o t e i n imino a c i d s a r e not i n c o r p o r a t e d i n t o p r o t e i n s t o produce s k e l e t a l a b n o r m a l i t i e s i n a way analogous t o c a n a v a n i n e , the n o n p r o t e i n homologue o f a r g i n i n e ( 8 ) . Some o t h e r mechanism must be i n v o l v e d . I n a s e r i e s o f experiments w i t h Spodoptera f r u g i p e r d a (7) v a r i o u s n u t r i t i o n a l i n d i c e s were c a l c u l a t e d a c c o r d i n g to Waldbaur ( 9 ) . We found t h a t C a l l i a n d r a l e a f m a t e r i a l , the t o t a l amino a c i d f r a c t i o n , and some o f the i n d i v i d u a l imino a c i d s d e p r e s s e d ECD ( e f f i c i e n c y o f c o n v e r s i o n o f d i g e s t e d food) and ECI ( e f f i c i e n c y o f c o n v e r s i o n o f i n g e s t e d food)


5. ROMEO & SIMMONDS


Table I I I .

Feeding DeterrentsfromCalliandra

65

A n t i f e e d a n t Index [ ( C - T ) / ( C + T ) % ] mean (sem)

Compound C4 C5 CC T4 T5 TT TC CT S-CEC

S. l i t t o r a l i s 52.9 (4.25)* -41.9 (21.6) -29.5 (15.4) 13.6 (15.6) 21.6 ( 5.6) -15.6 (25.6) -15.6 (26.6) 8.1 (12.4) 51.4 ( 1 5 . 9 ) *

H. v i r e s c e n s 38.2 (12.6)* -12.7 ( 5.6) -21.5 ( 4.6)

H. a r m i g e r a 41.5 ( 1 4 . 4 ) * -13.4 (12.6) -13.8 (15.7)

- 2.5 -21.4 8.6 37.8

- 8.4 (13.0) - 6.9 (21.6) 13.6 (12.9) 35.6 ( 1 2 . 3 ) *

(12.6) (21.6) (21.6) (12.6)*

C4 = c i s - 4 - h y d r o x y p i p e c o l i c a c i d ; C5 = c i s - 5 - h y d r o x y p i p e c o l i c a c i d ; CC = c i s - c i s - 4 , 5 - d i h y d r o x y p i p e c o l i c a c i d ; T4 = t r a n s - 4 h y d r o x y p i p e c o l i c a c i d ; T5 = t r a n s - 5 - h y d r o x y p i p e c o l i c a c i d ; TT = t r a n s - t r a n s - 4 , 5 - d i h y d r o x y p i p e c o l i c a c i d ; CT = c i s - t r a n s - 4 , 5 d i h y d r o x y p i p e c o l i c a c i d ; S-CEC = S - ( 3 - c a r b o x y e t h y l ) - c y s t e i n e . Concentration

100 ppm, η = 15-20

* S i g n i f i c a n t a c t i v i t y , ρ < 0.05

T a b l e IV.

W i l c o x e n matched p a i r s t e s t .

D e t e r r e n c e A c t i v i t y o f N o n p r o t e i n Amino A c i d s t o A p h i d F e e d i n g ( E x p r e s s e d as P e r c e n t D e c r e a s e of C o n t r o l s )

ίο"

5

2.7 19.4 8.3 10.2

ΙΟ"* 18.9 5.6 0// 20.5

C5 + CT C5 + PIP

14.7 5.9

8.8 11.7

C5 + CT + PIP C5 + CT + S-SEC PIP + CT + S-CEC

0# 19.3 22.5*

3.2 32.2* 35.4*

C5 + CT + PIP + S-CEC

38.5*

48.7*

Cone. (M)

Compound C5 CT PIP S-CEC

C5 = c i s - 5 - h y d r o x y p i p e c o l i c a c i d ; CT = c i s - t r a n s - 4 , 5 - d i h y d r o x y p i p e c o l i c a c i d ; PIP - p i p e c o l i c a c i d ; S-CEC - S - ( 3 - c a r b o x y e t h y l ) cysteine; * S i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l (Mann-Whitney t a i l e d , ρ < 0.025). // Number o f a p h i d s

f e e d i n g was g r e a t e r t h a n

t e s t , one

control.



66


(Table V I ) . RCR ( r e l a t i v e consumption r a t e ) and AD (approximate d i g e s t i b i l i t y ) were a f f e c t e d by the l e a f m a t e r i a l but not by the amino a c i d s . Thus the compounds behave l i k e p h y s i o l o g i c a l t o x i n s and a p p a r e n t l y are t a k e n up from the gut i n t o the hemolymph where they a d v e r s e l y a f f e c t m e t a b o l i s m . The s i m i l a r i t y of the imino compounds t o p o l y h y d r o x y a l k a l o i d s , which a r e known t o a c t as sugar c o m p e t i t o r s and i n h i b i t a v a r i e t y o f g l y c o s i d a s e enzymes i n i n s e c t s (1G), l e d us to t e s t t h e s e compounds a g a i n s t a number of enzymes. No a c t i v i t y of any of the mono o r d i h y d r o x y compounds was found ( F e l l o w s and Romeo, u n p u b l i s h e d data). A t r i h y d r o x y p i p e c o l i c a c i d r e c e n t l y i s o l a t e d from seeds of a n o t h e r legume, B a p h i a racemosa, however, w h i l e h a v i n g no e f f e c t on a l p h a and b e t a - g l u c o s i d a s e or mannosidase, i s a s p e c i f i c i n h i b i t o r o f human l i v e r b e t a - D - g l u c u r o n i d a s e and i d u r o n i d a s e ( Π - ) . The r e c e n t d e t e c t i o n o f a p r o b a b l e isomer o f t r i h y d r o x y p i p e c o l i c a c i d i n l e a v e s of s e v e r a l C a l l i a n d r a s p e c i e s i s of i n t e r e s t i n t h i s r e g a r d (Swain and M o r t o n , p e r s o n a l communication). In an attempt t o c h a r a c t e r i z e the mode o f a c t i o n of the d e t e r r e n t e f f e c t on the l a r v a e o f S^ l i t t o r a l i s , c o r r e l a t i o n s were made between the r e s u l t s o f e l e c t r o p h y s i o l o g i c a l r e c o r d i n g s from t a s t e s e n s i l l a and the r e s u l t s o f f e e d i n g b e h a v i o r s t u d i e s . Compounds were d i s s o l v e d i n sodium c h l o r i d e e l e c t r o l y t e (0.05M) to g i v e c o n c e n t r a t i o n s o f lOOOppm, lOOppm and lOppm. The m a x i l l a r y s t y l o c o n i c s e n s i l l a o f f i n a l s t a d i u m l a r v a e o f Spodoptera l i t t o r a l i s were s t i m u l a t e d w i t h t h e s e s o l u t i o n s u s i n g a s t a n d a r d t i p - r e c o r d i n g technique (12). To a l l o w comparison o f the e f f e c t o f a range o f compounds on a s i n g l e l a r v a , the l a t e r a l and m e d i a l s t y l o c o n i c s e n s i l l a o f each l a r v a were s t i m u l a t e d f o r 1 second s e q u e n t i a l l y w i t h 4-6 compounds, each a t t h r e e c o n c e n t r a t i o n s w i t h t h r e e r e p l i c a t i o n s per c o n c e n t r a t i o n . S u c c e s s i v e s t i m u l a t i o n s o f any one s e n s i l i u m were s e p a r a t e d by 2-3 m i n u t e s . The e l e c t r o l y t e , sodium c h l o r i d e , was used as a c o n t r o l and was a p p l i e d p e r i o d i c a l l y to make sure the p r e p a r a t i o n was not m a l f u n c t i o n i n g . The s o l u t i o n s can s t i m u l a t e 0 t o 4 o f the 4 c h e r o o s e n s i t i v e neurones i n each of the sensilla. The l e v e l o f s t i m u l a t i o n i s measured by c o u n t i n g the number of a c t i o n p o t e n t i a l s e l i c i t e d from the s e n s i l l u m i n the f i r s t second o f s t i m u l a t i o n . The r e s u l t s o f t h e s e a s s a y s a l l o w us t o attempt t o c o r r e l a t e n e u r a l i n p u t w i t h the r e s u l t i n g b e h a v i o r a l o u t p u t . The n e u r a l i n p u t can be c o r r e l a t e d w i t h s h o r t term b e h a v i o r , the d u r a t i o n o f the f i r s t meal on the t r e a t m e n t d i s c , or w i t h the l o n g e r term b e h a v i o r a l r e s p o n s e s , the amount o f time spent f e e d i n g on the treatment d i s c w i t h i n the f i r s t two h o u r s or the v a l u e o f the A n t i f e e d a n t Index. In p r e v i o u s s t u d i e s n e u r a l i n p u t from the m a x i l l a r y s t y l o c o n i c s e n s i l l a has c o r r e l a t e d s i g n i f i c a n t l y w i t h f e e d i n g b e h a v i o r (13,14,15). A l t h o u g h the i m i n o and s u l p h u r amino a c i d s s t i m u l a t e 1-2 neurons i n b o t h the m e d i a l and l a t e r a l m a x i l l a r y s t y l o c o n i c s e n s i l l a , t h i s response does not c o r r e l a t e w i t h e i t h e r the d u r a t i o n of the f i r s t c o n t a c t w i t h the treatment d i s c , time spent f e e d i n g on the treatment d i s c i n the f i r s t two h o u r s , or the A n t i f e e d a n t Index. T h i s s u g g e s t s t h a t t h e s e s e n s i l l a a r e not r e s p o n s i b l e f o r any n e u r a l i n p u t t h a t might be a s s o c i a t e d w i t h the a n t i f e e d a n t a c t i v i t y r e c o r d e d w i t h C4 and S-CEC. What we do not know, however, i s i f t h e s e compounds i n t e r a c t w i t h s u c r o s e p r e s e n t on the d i s c s used i n the b e h a v i o r a s s a y . I n t e r a c t i o n s between a l l e l o c h e m i c s and s u c r o s e


5.


T a b l e V.

T o x i c i t y o f N o n p r o t e i n Amino A c i d s t o A p h i d s ( E x p r e s s e d as P e r c e n t D e c r e a s e i n Number A l i v e A f t e r 5 days Compared t o C o n t r o l s )

C5 CT PIP S-CEC

l(f 0# 19.3 5.3 21.3

ΙΟ"* 19.8 30.2* 5.3 22.9

C5 + CT C5 + PIP

16.3 3.4

9.5 14.6

C5 + CT + PIP C5 + CT + S-CEC PIP + C5 + S-CEC PIP + CT + S-CEC

38.3* 59.4* 55.6* 60.1*

37.6* 68.4* 51.8* 60.1*

C5 + CT + PIP + S-CEC

68.8*

82.4*

Cone. (M)

Compound


67


5

Compound a b b r e v i a t i o n s - same as T a b l e IV. *Significantly different t a i l e d , ρ < 0.025).

from c o n t r o l

# Number o f a p h i d s was g r e a t e r than

Table V I .

control.

E f f e c t o f C a l l i a n d r a l e a f m a t e r i a l and amino a c i d s on n u t r i t i o n a l p h y s i o l o g y o f S p o d o p t e r a f r u g i p e r d a (Adapted f r o m Shea, 1987)

Treatment Control 2.5% C. haematocephala Control Amino A c i d s 0.1% TT 0.1% C5 0.5% T5

(Mann-Whitney t e s t , one

(100% P l a n t

Equivalency)

ECI .23 .14*

AD .48 .40*

ECD .54 .36*

.27 .27 .23* .26 .25

.45 .49 .49 .45 .49

.66 .57* .52* .59 .53*

ECI = e f f i c i e n c y o f c o n v e r s i o n o f i n g e s t e d f o o d ; AD = a p p r o x i m a t e d i g e s t i b i l i t y ; ECD = e f f i c i e n c y o f c o n v e r s i o n o f d i g e s t e d f o o d . TT = t r a n s - t r a n s - 4 , 5 - d i h y d r o x y p i p e c o l i c a c i d ; C5 = c i s - 5 h y d r o x y p i p e c o l i c a c i d ; T5 = t r a n s - 5 - h y d r o x y p i p e c o l i c a c i d . * S i g n i f i c a n t l y d i f f e r e n t from c o n t r o l f o r unequal sample s i z e , ρ < 0.05)

f

(ANOVA and T u k e y s

test



68

have r e s u l t e d i n a d e c r e a s e i n t h e f i r i n g r a t e o f b o t h t h e m a x i l l a r y s t y l o c o n i c s e n s i l l a , when compared t o t h e response o b t a i n e d from e i t h e r t h e a l l e l o c h e m i c o r s u c r o s e a p p l i e d s e p a r a t e l y (1^,14). I n such c a s e s , t h e d e c r e a s e i n n e u r a l i n p u t has been c o r r e l a t e d w i t h an increase i n antifeedant a c t i v i t y . T h i s remains t o be i n v e s t i g a t e d i n t h i s case.


Conclusion The r e s u l t s o f o u r a p h i d experiments emphasize t h e importance o f l o o k i n g a t c o m b i n a t i o n s o f compounds when r u n n i n g b i o a s s a y s t o t e s t for deterrent or toxic e f f e c t s . C l e a r l y w i t h b o t h t h e imino and s u l p h u r amine a c i d s we a r e l o o k i n g a t i n d i v i d u a l compounds o f marginal i n s e c t i c i d a l a c t i v i t y . I n c o n c e r t , however, they a r e shown t o be c o n s i d e r a b l y more e f f e c t i v e . W h i l e such s y n e r g i s m i s c e r t a i n l y n o t a new i d e a i n c h e m i c a l e c o l o g y (16) , we perhaps need to g i v e i t renewed a t t e n t i o n i n o u r t e s t s f o r b i o l o g i c a l a c t i v i t y . There a r e p r o b a b l y many such a r r a y s o f e f f e c t i v e d e t e r r e n t s h e r e t o f o r e d i s m i s s e d as u n i m p o r t a n t .

Literature Cited 1.

Calliandra: A Versatile Small Tree for the Humid Tropics; National Academy Press, Washington, DC, 1983; pp 3-7. 2. Uribe, B.; Ospina, Α.; E. Forero. Programa Interciencia de Recursos Biologicos; Guadalupe LTDA, Bogota, Colombia, 1984; pp 104-107. 3. Romeo, J . T.; Swain, L. Α.; Bleecker, A. B. Phytochemistry 1983, 22, 1615-1617. 4. Swain, L. Α.; Romeo, J.T. Phytochemistry 1988, 27, 397-399. 5. Romeo, J. T.; Swain, L. A. J. Chem. Ecol. 1986, 12, 2089-2096. 6. Romeo, J.T. Biochem. Syst. & Ecol. 1984, 12, 293-297. 7. Shea, C. S. MS Thesis, University of South Florida, Tampa, F l , 1987. 8. Dahlman, D. L . ; Rosenthal, G. A. Comp. Biochem. Physiol. A. 1975, 51A, 33-36. 9. Waldbaur, G. P. Adv. Insect Physiol. 1968, 5, 229-288. 10. Evans, S. V.; Fellows, L. E.; Shing, T. Κ. M.; Fleet, G. W. J . Phytochemistry 1985, 24, 1953-1955. 11. Cenci di Bello, I.; Dorling, P.; Fellows, L . ; Winchester, B. FEBS Lett. 1984, 176, 61-64. 12. Blaney, W. M. J . Exp. Biol. 1974, 60, 275-293. 13. Simmonds, M. S. J.; Blaney, W. M. In. Proc. 2nd. Int. Neem Conf. 1984, pp.163-18. 14. Elaney, W. M.; Simmonds, M. S. J.; Ley, S.U.; Katz, R. B. An. Phys. Entom. 1987, 12, 281-291. 15. Blaney, W. M.; Simmonds, M. S. J.; Ley, S. V.; Jones, P.S. Entomol. Exp. Appl. 1988, 46, 267-274. 16. Berenbaum M. In Chemically Mediated Interactions between Plants and Other Organisms; Cooper-Driver, G. Α.; Swain, T.; Conn, Ε. E. Eds. Recent Advances in Phytochemistry Vol. 19; Plenum Press, New York, pp. 139-169. RECEIVED November 2, 1988


Insecticides of Plant Origin - American Chemical Society

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