Isolation of Phytoecdysones as Insect Ecdysis Inhibitors and Feeding

19 Isolation of Phytoecdysones as Insect Ecdysis Inhibitors and Feeding Deterrents ISAO K U B O and J A M E S A . K L O C K E

Downloaded by FUDAN UNIV on March 15, 2017 | http://pubs.acs.org Publication Date: January 20, 1983 | doi: 10.1021/bk-1983-0208.ch019

University of California, Division of Entomology and Parasitology, College of Natural Resources, Berkeley, CA 94720

Phytoecdysones, due t o their e f f e c t s on the behavior and the development of certain species o f i n s e c t s , appear t o be components of multichemical defensive s t r a t e g i e s found in some i n s e c t - r e s i s t a n t p l a n t species. Observations in nature obviate the f a c t that c e r t a i n p l a n t species and cultivars are more r e s i s t a n t t o i n s e c t a t t a c k 1~3 than are others. Ajuga remota (Labiatae) is an example of this. A survey of a Kenyan savannah f o l l o w i n g a l o c u s t a t t a c k revealed that the only v e g e t a t i o n t o s u r v i v e the a s s a u l t was A. remota4. In order t o t e s t f o r chemical f a c t o r s i n v o l v e d i n this observed r e s i s t a n c e , e x t r a c t s of A. remota f o l i a g e were i n c o r p o r a ted i n t o a r t i f i c i a l d i e t s optimized f o r s e v e r a l economically important pest i n s e c t s ( F i g . 1 ) 5 . B r i e f l y , a methanolic e x t r a c t was d i s s o l v e d i n solvent and added t o a n o n - n u t r i t i v e f i l l e r ( a - c e l l u l o s e ) , evaporated t o dryness, and added t o the components of a meridic a r t i f i c i a l d i e t , i n c l u d i n g s o l i d n u t r i e n t s ( c a s e i n , sucrose, wheat germ, Wesson s a l t s ) , vitamins (C and B-complex), and 4% agar. Newly-hatched larvae of the pink bollworm, Peetinophora gossypiella and of the f a l l armyworm, Spodoptera frugiperda were placed s i n g l y on p o r t i o n s of the d i e t i n p l a s t i c v i a l s . A d d i t i o n a l bioassays were conducted w i t h the silkworm, Bombyx moriy by i n c o r p o r a t i n g d i s s o l v e d A* remota e x t r a c t s d i r e c t l y i n t o d r i e d mulberry powder (Nihon Nosan), evaporating the solvent t o dryness, and adding a 2% agar s o l u t i o n . A n a l y s i s of the t e s t i n s e c t s f e d the A. remota e x t r a c t s r e vealed a developmental d i s r u p t i o n i n which the i n s e c t s died i n the pharate c o n d i t i o n f o l l o w i n g i n i t i a t i o n of molting ( a p o l y s i s ) , but before completion of molting ( e c d y s i s ) ( F i g . 2 - 4 ) 6 . Insect molting c y c l e i s i n i t i a t e d when the c u t i c u l a r e p i t h e l i u m separ a t e s from the o v e r l y i n g c u t i c l e i n the process of a p o l y s i s . The molting c y c l e i s terminated, upon the completion of c u t i c l e synt h e s i s , by h y d r o s t a t i c expansion of the new c u t i c l e during the process of e c d y s i s . The A* remota e x t r a c t apparently upset the 0097-6156/ 83/0208-0329$06.00/0 © 1983 American Chemical Society

Hedin; Plant Resistance to Insects ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

330

PLANT RESISTANCE TO INSECTS


Sample Solution

5 ug ponasterone A were induced t o i n i t i a t e m o l t i n g ( a p o l y s i s ) , but were unable t o comp l e t e t h i s molt due t o an i n h i b i t i o n of e c d y s i s . Thus, a l l of these ponasterone A-treated l a r v a e d i e d i n the pharate c o n d i t i o n . 7

1

y

10

8



334

Table 1 . E f f e c t s o f 5 phytoecdysones on growth and development o f pink bollworm l a r v a e . Amount i n d i e t (ppm)

Compound

35


fi-ecdysone

Cyasterone

Effect

ED^Q

50

E I

25

E D ^

40

Ponasterone A

Ajugasterone C

E I

1

E

2

EI

E

14

45

D

5

9

5

5 0 9 5

5 0

EI

430

Ajugalactone

D

9

9 5

ED^^

*

EI

Values are based on three o r more r e p l i c a t e s , each of which c o n s i s t e d o f 3 0 o r more neonate pink b o l l worm

assayed

effective EI82% o f treated compared t o c o n t r o l .

larvae

t o a p o l y s i s f o r > 72 hr

(P) i n d i c a t e s 10 yg of B-ecdysone a l s o r e s u l t e d i n a promoted a p o l y s i s , but depending upon c o n c e n t r a t i o n and the exact age of the t r e a t e d f o u r t h i n s t a r l a r v a e , a v a r i a b l e number of the t r e a ted l a r v a e were able t o complete m o l t i n g and e s s e n t i a l l y recover. Cyasterone i n j e c t i o n s >10 pg, i n g e n e r a l , caused a delay ( a n t i ecdysone) of m o l t i n g of the t r e a t e d f o u r t h i n s t a r l a r v a e t o t h e f i f t h instar. This 'antiecdysone e f f e c t of cyasterone, at i t s most severe, r e s u l t e d i n p r o t h e t e l y (precocious development) of a small % of the t r e a t e d l a r v a e . In order t o examine a n a t o m i c a l l y the newly-synthesized head of f o u r t h i n s t a r l a r v a e undergoing m o l t i n g c y c l e f a i l u r e , the adhering e x u v i a l head capsule was c a r e f u l l y removed w i t h f o r c e p s . This procedure revealed the morphological d i s r u p t i o n of the f e e d ing apparatus ( F i g . 7). The f i f t h i n s t a r head capsule was compressed by the adhering f o u r t h i n s t a r head capsule, which r e s u l ted i n the pushing forward of the m a x i l l o l a b i a l - h y p o p h a r y n g e a l complex (mc) such that the mandibles could not f u l l y c l o s e . In order t o study how i n j e c t e d (or ingested) phytoecdysone causes an i n h i b i t i o n i n e c d y s i s , s e v e r a l biochemical parameters of l a r v a l m o l t i n g f l u i d were analysed, i n c l u d i n g phenoloxidase a c t i v i t y , t o t a l p r o t e i n , and t o t a l a s c o r b i c a c i d (Table 3 ) . M o l t i n g f l u i d may be i m p l i c a t e d i n the i n h i b i t i o n of e c d y s i s since e i t h e r t o t a l removal of the m o l t i n g f l u i d (unpublished) o r a simple d e l e t i o n of a s c o r b i c a c i d from the m o l t i n g f l u i d of Spodoptera littoralis (Navon, 1 9 7 8 ) ( f e d w i t h no phytoecdysone) d u p l i c a t e d the phytoecdysone-induced i n h i b i t i o n of e c d y s i s . The a n t i o x i d a n t a s c o r b i c a c i d has been hypothesized t o c o n t r o l phenol o x i d a s e hardening of the newly-synthesized c u t i c l e before i t s h y d r o s t a t i c expansion. I n a d d i t i o n , B-ecdysone i s known t o a c t i vate the enzyme which c a t a l y s e s the s y n t h e s i s of a phenoloxidase from i t s proenzyme ^. Nevertheless, a s c o r b i c a c i d l e v e l s i n both the m o l t i n g f l u i d and the hemolymph of phytoecdysone-treated l a r v a e are comparable to those l e v e l s found i n untreated l a r v a e . In a d d i t i o n , phenoloxidase a c t i v i t y , as measured w i t h c a t e c h o l s u b s t r a t e , i s a c t u a l l y l e s s i n phytoecdysone-induced l a r v a e as compared t o c o n t r o l larvae. These negative r e s u l t s do not e l i m i n a t e the involvement i n ecdysis i n h i b i t i o n o f other biochemical parameters i n the m o l t i n g f l u i d , i . e . c h i t i n a s e , protease, but do seem t o i n d i c a t e that premature phenoloxidase-catalysed c u t i c l e hardening i s not the cause f o r phytoecdysone-induced f a i l e d e c d y s i s . More d e t a i l e d data w i t h B. mori showed that the e f f e c t s o f ingested B-ecdysone i n c l u d e d , besides an i n h i b i t i o n of e c d y s i s , death without m o l t i n g , death f o l l o w i n g completion of promoted m o l t i n g , and an i n h i b i t i o n i n growth w i t h no e f f e c t on m o l t i n g . These v a r i o u s e f f e c t s are dependent upon the c o n c e n t r a t i o n of exogenous B-ecdysone, the p r e c i s e developmental stage of the treated l a r v a e , and the d u r a t i o n of exposure. Concentrations of B-ecdysone 5 0 ppm i n a r t i f i c i a l d i e t i n 1


337

11

1

>


338


Table 3.

Comparison ascorbic acid

of

acid

levels

following

phenoloxidase levels

i n the molt

i n the

apolysis

activity,

hemolymph

protein,

fluid

and

ascorbic

approximately

to the 5th i n s t a r .

and

36

B. mori

hr

larvae

were i n j e c t e d per os at 4th i n s t a r 2nd day w i t h 10 u l Downloaded by FUDAN UNIV on March 15, 2017 | http://pubs.acs.org Publication Date: January 20, 1983 | doi: 10.1021/bk-1983-0208.ch019

30% aq/EtOH alone o r w i t h 10 y l 30% aq/EtOH + phytoecdysone.

25 larvae

were used/treatment/parameter

and

analyzed.

Orally

A

4 7 Q

/min/

ug A s c o r b i c

ug A s c o r b i c

ug P r o t e i n /

applied

u l molt

acid/ul

u l molt

treatment

fluid

molt f l u i d

hemolymph

fluid

Control

0.031 +

0.05

0.13

0.06

0.12

acid/ul

16.0

(solvent o n l y ) 0.012

30 ug

0.008

3-Ecdysone

+

(in solvent)

0.005

10 ug

0.006

Ponasterone A

15.0

(in solvent)



19.

KUBO

AND KLOCKE

Isolation of

Phytoecdysones

339

duced premature m o l t i n g i n 5. mori and r e s u l t e d i n 100% m o r t a l i t y . Much of t h i s m o r t a l i t y , however, occurred not as a r e s u l t of an i n h i b i t i o n of e c d y s i s , but a c t u a l l y a f t e r molting had been completed. I n f a c t , w h i l e the lower c o n c e n t r a t i o n s o f B-ecdysone r e s u l t e d i n ne cannot be e n t i r e l y exp l a i n e d by an i n h i b i t i o n of e c d y s i s . At 25 ppm B-ecdysone, m o l t i n g i s delayed (Table 5) so that 88% of the exposed l a r v a e d i e during the second i n s t a r ( t h a t i s , before m o l t i n g o c c u r s ) . Doses > 6.25 ppm but 25 ppm) had a high t i t re of ecdysone b u i l t i n the hemolymph, a t i t r e which could not be metabolized o r excreted r a p i d l y enough t o prevent hormonal imbalance r e s u l t i n g i n molting promot i o n and death. Larvae fed lower c o n c e n t r a t i o n s (< 25 ppm) o f B-ecdysone grow more slowly than c o n t r o l and molt l a t e r than control. P o s s i b l y the ecdysone a t these lower c o n c e n t r a t i o n s induced metabolism of both trie exogenous and the endogenous ecdysone such that there was a delay i n a p o l y s i s . H i k i n o et at. ( 1 9 7 5 ) showed that the c a t a b o l i c a c t i v i t y on B-ecdysone o f B* mox*i v a r i e d during the course of i t s growth and development. This i s i l l u s t r a t e d i n Tables 2 and 6 i n which i t can be seen that the l a r v a e are more s e n s i t i v e t o e i t h e r i n gested (Table 6) o r i n j e c t e d (Table 2) 3-ecdysone d u r i n g t h e e a r l i e r phase o f the f o u r t h i n s t a r . An a d d i t i o n a l bioassay w i t h a pest aphid species was conducted t o t e s t f o r a feeding d e t e r r e n t e f f e c t o f d i e t a r y phytoecdys o n e s ^ ( F i g . 8 ) . One of s e v e r a l phytoecdysones was d i s s o l v e d d i r e c t l y i n t o an aqueous d i e t optimized f o r maximal aphid f e e d i n g . The c o n t r o l aqueous d i e t c o n s i s t e d of vitamins (C and Bcomplex), sucrose, amino a c i d s , t r a c e metals, s a l t s , c h o l e s t e r o l , brought t o pH 8.7 w i t h K3PO4. The aqueous d i e t was placed i n t o poly-ethylene v i a l caps and each o f these caps were f i t t e d i n t o c i r c u l a r holes punched i n t o p l a s t i c snap-on l i d s f o r p o l y styrene catsup cups (1 o z ) . Between 50-100 Biotype C greenbugs, Schizaphis graminim , an important pest on sorghum (and other economically important g r a i n s ) i n the midwestern U.S., were t r a n s f e r r e d from sorghum p l a n t s i n t o each of the 1 oz catsup cups which were immediately f i t t e d w i t h the d i e t cap-containing snapon l i d s . A f t e r 24 h r s a t room temperature the no. of aphids f e e d i n g / t o t a l no. aphids was determined f o r each treatment. 13

1


340


Table 4.

E f f e c t s of fi-ecdysone on the l a r v a l development a


of second i n s t a r Borribyx mori

Concen-

%

tration

Development t o

% of t o t a l

mortality

occurring as:

Total

i n diet

3rd

4th

mortality

2nd

Ecdysis

3rd

(ppm)

instar

instar

%

instar

inhibition

instar

100

28

0

100

72

0

28

50

28

0

100

56

16

28

25

8

0

100

88

4

8

12-5

63

50

51

41

33

25

73

69

31

26

61

13

3.125

100

96

4

0

0

100

Control

100

96

4

0

0

100

6.25

a) 25 second i n s t a r f i r s t day larvae/treatment


19.

KUBO

AND KLOCKE

341

Isolation of Phytoecdysones

Table 5. E f f e c t s of B-ecdysone on the l a r v a l developmental p e r i o d of second i n s t a r Bombyx mori


Concentration

Average Period (days) t o

in diet

Molted

(ppm)

2nd I n s t a r

100

b

3.0(7) '

50

3.9(ll)

25

8.0(2)

12.5

Ecdysed

Ecdysed

c

c

c

3rd I n s t a r

4th I n s t a r

4.1(7)

c

4.9(7)

c

-

Promotion

9.5(2)

c

-

Molting

Molting

6.9(15)

c

14.3(10)

6.0(19)

c

14.3(18)

6.4(19)

c

6.25

6.0(24)

c

3.125

5.0(25)

6.0(25)

13.7(24)

Control

4.9(25)

5.9(25)

14.0(24)

a) 25 second i n s t a r f i r s t day larvae/treatment.

b) Figures i n p a r e n t h e s i s show the number of l a r v a e a l i v e out of the o r i g i n a l 25 second i n s t a r f i r s t day l a r v a e .

c) S i g n i f i c a n t d i f f e r e n c e

from

control

a t P=0.001

by Mann-Whitney U t e s t .


Delay

342


Aqueous Sample S o l u t i o n


Akey Diet Amino a c i d s , Sucrose, B-vitamins Vitamin C, S a l t s , Trace Metals Cholesterol ajusted to pH 8.7 350 v l 1.5 cm ID polyethylene v i a l cap Parafilm

Catshup cup l i d w i t h inserted d i e t cap

50-100 Biotype C aphids i n 1 oz. catshup cup

Observation o f Number Feeding a f t e r 24 h

Figure 8.

Antifeedant bioassay for the greenbug, Schizaphis graminum-


19.

KUBO

Isolation of

AND KLOCKE

343

Phytoecdysones

Table 6. E f f e c t s of varying no- consecutive days feeding on d i e t a r y fi-ecdysone by second i n s t a r B. movi larvae


Days of treatment w i t h fi-ecdysone f o l l o w i n g

Concentration of

molt t o 2nd i n s t a r

d i e t a r y fi-ecdysone (ppm)

a

of B. moH l a r v a e )

100

l s t - 5 t h days i n c l u s i v e 1st day only

1

50

25

12.5

6.25

0.20

0.22 j 0.30

0.48

0.84

0.61

0.67

0.66

0.69

0.86

2nd day only

I 0.58

0.66

0.67

0.74

0.88

3rd day only

0.68

0.72

0.75

0.80

0.86

4th day only

0.83

0.78

0.86

0.93 0.90

a)

25 second i n s t a r f i r s t day larvae/treatment

b)

No. i n d i c a t e weight

ratio

1

of t r e a t e d l a r v a e / c o n t r o l

l a r v a e on the f i f t h day of second i n s t a r .

i n d i c a t e s treatment a f f e c t i n g i n h i b i t i o n of m o l t i n g t o the t h i r d i n s t a r .

i n d i c a t e s treatment a f f e c t i n g promotion of molting t o the t h i r d i n s t a r .



344


Appropriate c o n t r o l s , which c o n s i s t e n t l y r e s u l t e d i n > 90% feedi n g , were then used to compare to each of the treatments i n order to determine ED50 values f o r each of the t e s t e d phytoecdysones. ED50 i s the e f f e c t i v e dose f o r 50% feeding compared to c o n t r o l . From Table 7 i t can be seen that ajugasterone C i s more than 1 0 - f o l d more potent than fi-ecdysone, and more than 3 0 - f o l d more potent than cyasterone, as a feeding d e t e r r e n t to S. graminum when i n c o r p o r a t e d i n t o the a r t i f i c a l d i e t of t h i s b e h a v i o r a l b i o assay. Although ingested phytoecdysones do have a potent and unique hormonal a c t i v i t y a g a i n s t s u s c e p t i b l e species l i k e the silkworm and the pink bollworm, other i n s e c t species are unaffected by d i e t a r y phytoecdysones. For example, Heliothis complex fed more than 3000 ppm phytoecdysones i n a r t i f i c i a l d i e t show no obvious morphological or developmental changes. However, other chemicals besides phytoecdysones contained i n ' r e s i s t a n t ' p l a n t s l i k e A. remota and P. gracilior do have a v a r i e t y of e f f e c t s a g a i n s t Heliothis and other i n s e c t s p e c i e s . A . remota e x t r a c t s have y i e l d e d 6 d i t e r p e n e s causing a n t i f e e d a n t and i n s e c t i c i d a l a c t i v i •.yl-3,15^ while s e v e r a l i n s e c t i c i d a l n a g i l a c t o n e s coupled to an a n t i f e e d a n t a c t i v i t y as w e l l as two g r o w t h - i n h i b i t i n g b i s f l a vones have been i s o l a t e d from P. gracilior foliage^. Such multicomponent defensive s t r a t e g i e s , as those e l u c i d a t e d i n A* remota and i n P. gracilior, may be more the r u l e than the exception i n r e s i s t a n t p l a n t c u l t i v a r s . The e l u c i d a t i o n of these s t r a t e g i e s , p a r t i c u l a r l y the chemical aspects of them, i s important f o r an understanding of e c o l o g i c a l and e v o l u t i o n a r y aspects of host p l a n t r e s i s t a n c e . In a d d i t i o n , the mechanistic understanding of host p l a n t r e s i s t a n c e may have economical i m p l i c a t i o n s i n that ' r e s i s t a n c e ' chemicals may be bred i n t o crop p l a n t s , or they may be e x t r a c t e d from one p l a n t species and a p p l i e d d i r e c t l y to another economically important p l a n t s p e c i e s , or they may serve as l e a d i n g structui»2S i n s y n t h e t i c p e s t i c i d e research. The r o l e of phytoecdysones i n t h i s scheme i s that of an important component i n a r a t h e r complex defensive s t r a t e g y of some p l a n t s . T h e i r presence i n p l a n t s probably serves a l i m i t e d , yet important, p r o t e c t i v e r o l e . For example, c o t t o n b o l l s bred w i t h s e v e r a l ppm of ponasterone A would very l i k e l y be r e s i s t a n t to a t t a c k by pink bollworm. In summary, then, whether a c t i n g alone or i n c o n j u n c t i o n w i t h other chemicals, the unique and potent p h y s i o l o g i c a l , biochemic a l , and morphological e f f e c t s induced by the phytoecdysones conf e r s an i n t e g r a l r o l e f o r them i n host p l a n t r e s i s t a n c e .


KUBO AND KLOCKE

Isolation

of

Phytoecdysones


Table 7. Feeding deterrency of three phytoecdysones on greenbug, Sohizaphis

Compounds

E

D

5 0

graminurft

*

B-ecdysone

650

Cyasterone

2000

Ajugasterone C

a) Biotype

C o f 5. graminum

n

diet)**

62

from a mixed

p o p u l a t i o n i n a 2 4 h no-choice bioassay.

b) E D ^ Q i s the e f f e c t i v e dose f o r 50% feeding compared t o c o n t r o l .


346

PLANT

RESISTANCE

TO

INSECTS

Acknowledgement I n s e c t s were k i n d l y supplied by the agencies of the USDA i n B r o w n s v i l l e , Tx; Phoenix, Az; and T i f t o n , Ga. The authors thank J . DeBenedictis f o r h i s help w i t h e l e c t r o n micrographs and D. Dreyer and K. Jones f o r t h e i r help w i t h the aphid bioassay. Authentic samples of phytoecdysones were g i f t s from P r o f e s s o r T. Takemoto ;and P r o f e s s o r K. Nakanishi.


References 1. Kubo, I . ; Lee, Y. W.; Balogh-Nair, V.; N a k a n i s h i , K.; Chapya, A. J. Chem. Soc. Chem. Commun. 1976, 949. 2. Kubo, I . ; Kido, M.; Fukuyama, Y. J. Chem. Soc. Chem. Commun., 1980, 897. 3. Kubo, I . ; K l o c k e , J . A.; Miura, I . ; Fukuyama, Y. J. Chem. Soc. Chem. Commun., 1982, 618. 4. Kubo, I . Science Year 1982, World Book-Childcraft International, Chicago, 1981, 126. 5. Chan, B. G.; Waiss, A. C. Jr.; S t a n l e y , W. L.; Goodban, A. E. J. Econ. Entomol., 1978, 71, 366. 6. Kubo, I . ; Klocke, J. A.; Asano, S. Agric. Biol. Chem., 1981, 45, 1925. 7. Kubo, I . ; Klocke, J . A.; Asano, S., in p r e p a r a t i o n . 8. Kubo, I . ; Ganjian, I . ; Klocke, J . A., in p r e p a r a t i o n . 9. Kubo, I . ; K l o c k e , J . A., in p r e p a r a t i o n . 10. Hostettmann, K. Planeta Medica, 1980, 39, 1. 11. Navon, A. J. Insect Physiol., 1978, 24, 39. 12. Chapman, R. F. "The I n s e c t s , S t r u c t u r e and Function, American E l s e v i e r P u b l i s h i n g Company, New York, 1971, 700. 13. H i k i n o , H.; Ohizumi, Y.; Takemoto, T. J. Insect Physiol., 1975, 21, 1953. 14. Dreyer, D. L.; Reese, J . C.; Jones, K. C.J.Chem.Ecol., 1981, 21, 273. 15. Kubo, I . ; N a k a n i s h i , K. Host Plant Resistance to Pests, ACS Symposium S e r i e s 62, American Chemical S o c i e t y , Washington, D.C., 1977, 165. RECEIVED

September 24, 1982


Isolation of Phytoecdysones as Insect Ecdysis Inhibitors and Feeding

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