Insect Antifeedant Terpenoids in Wild Sunflower - ACS Symposium

30 Insect Antifeedant Terpenoids in Wild Sunflower A Possible Source of Resistance to the Sunflower Moth 1,4

51,

1

JONATHAN GERSHENZON , MARYCAROL ROSSITER, TOM J. MABRY, CHARLIE E. ROGERS , MICHAEL H. BLUST, and THEODORE L. HOPKINS 2,6

3

3

1

Departments of Botany and Zoology, University of Texas, Austin, TX 78713

2

Conservation and Production Research Laboratory, Agricultural Research Service, U . S .

Downloaded by CORNELL UNIV on May 25, 2017 | http://pubs.acs.org Publication Date: April 26, 1985 | doi: 10.1021/bk-1985-0276.ch030

Department of Agriculture, Bushland, TX 79012 3

Department of Entomology, Kansas State University, Manhattan, KS 66506

Sunflower (Helianthus annuus) cultivation in the United States is frequently limited by the severity of insect damage. However, many wild Helianthus species are resistant to the major insect pests of cultivated sunflower. This resistance has been suggested to have a chemical basis. We found high concentrations of sesquiterpene lactones and diterpenes in glandular hairs on several resistant wild species of Helianthus and demonstrated that these compounds were toxins and antifeedants towards some major sunflower insect pests. Experiments were conducted on the southern armyworm (Spodoptera eridania), the migratory grasshopper (Melanoplus sanguinipes) and the sunflower moth (Homoeosoma electellum). Of particular interest was the presence of terpenoids on the portions of the flower immediately adjacent to the pollen. The early larval stages of the sunflower moth, the most destructive insect pest of cultivated sunflower in the United States, feed principally on pollen. Examination of several cultivated lines of sunflower showed that these had lower densities of glandular hairs than the wild species. Thus, increased resistance to sunflower moth predation might be achieved simply by breeding for an increased density of glandular hairs on floral parts surrounding the pollen.

4

Current address: Institute of Biological Chemistry, Washington State University, Pullman, W A 99164.

5

Current address: Department of Entomology, Pennsylvania State University, University Park, P A 16802.

6

C u r r e n t address: Insect Biology and Population Management Research Laboratory, Agricultural Research Service, U . S . Department of Agriculture, Tifton, G A 31793.

0097-6156/85/0276-0433$06.00/0 © 1985 American Chemical Society Hedin et al.; Bioregulators for Pest Control ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

BIOREGULATORS FOR PEST CONTROL

434

The

environmental

synthetic natural

products

best-studied levels

of

subject

and

economic

i n s e c t i c i d e s have to

toxic

to

control

approach of or

insect

insect

this

pests

type

antifeedant attack

to

damage

(1).

Our

suggest

that

plant-produced

contribution

drawbacks

is

the

of

increasing

to

scale

of

sunflower

resistance

may

to

of

plant

Perhaps for

in

the

greater

plant

resistance

products

use

using

breed

products

their

with

ways

crop plants.

natural

natural

large

on

simply

increase

investigations

towards

to

focused attention

parts

to

insect

(Helianthus

annuus)

make

insect

a

significant

damage

in

this

crop. Sunflower crops

in

s o i l ,


in

tion

of

their

has

further

new

problems

over of

are

insect

feed

on

in

of

major is

no

obvious

might

account

this

isolated results

of

discuss

the

insect

pest

describe damage

to

resistance greenhouse chemically were

and

to

f i e l d to

be

terpenoid

diterpenes.

diterpenes diterpenes

and in

We

have

most

have

fifty

terpenoid

of

Because plants

prédation,

in

the

the

constituents

and

present

compounds. the

Then

we to

destructive

States

in

the

resistance

most

United

exploited

species

t r i a l s a

(4), a n d

reducing

Helianthus

of

small

group

now high

the two

(13-25). from

began

insect

17

of

Both

our

parts

species of

by

which

The

major

these

plants

sesquiterpene species

in

studies

resistant.

aerial

shown some

sunflower

Helianthus

types:

studied levels

isolated

of

insect

of

have

cultivated We

(6-12).

compound o f have

of

pests

especially

these

been

of

moiety

the in

12 a n d

6 or

lactones

are

Asteraceae, these

methylene-γ-lactone atoms

in

some

of

lactones

Helianthus

sesquiterpene

lactones

and

sesquiterpene

lactones

and

species

of

Helianthus

by

other

(26-44).

members

lactone

insect

Helianthus these

be

to

Helianthus

insect

found

of

Sesquiterpene many

the

major

of

secondary metabolites be

chemically

workers

might

to

resistant

to

terpenoids

sunflower

adapt

sunflowers

(6-12).

these

(4).

because

c h e m i c a l b a s i s (12).

the

of

implicates

species

investigating

lipophilic

a

oil

therefore to

be r e s i s t a n t

in

(3).

sunflower.

of

the

reported

proved and

half

to

review

of

States

wild

of

limiting

(5) a n d

of

immunity

the

years

20

yield

varieties

feature

of

of

c u l t i v a -

factors

opportunity

(Homoeosoma e l e c t e l l u m ) ,

from w i l d

Approximately

shown to

species

information

last

United

number

bioassays with

which

cultivated

Terpenoids

a

relative

wild

the

o i l

range

concentrations

America

cultivated

f i r s t

the

had the

r e s i s t a n c e has

we

cultivated

this

in

wide

sunflower

depressing the

However,

this

in

vegetable

a

because

high

principal

North

been

to and

(2)

very

serious have

their

insect

moth

of

how

and to

the

major

States,

the

morphological for

evidence

sunflower

of

native

have

resistant

some

one

country

of

chapter,

from

with

United

hundred-fold

is

(4).

suggested that In

the

is

pests

which was

the

sunflower

Helianthus)

insect

there it

a

this

sunflowers

(species the

taxa

In

world's

adaptability

conditions

particularly

genus Helianthus

many

the

varieties

(3).

damage

cultivation

Insect

of its

water

hybrid

achenes

insect

one

because of

and

increased

Extensive

the

become

years

temperature

development oil

has

recent

8 on

with the

secondary metabolites

composite

compounds

function

12 a n d

typical

the

the

is

family

usually

lactone

present

bridge

sesquiterpene

(45,

46). as

joining

skeleton

(see

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

an

of The α

-

carbon Figure

30.

GERSHENZON ET AL.

Insect Antifeedant Terpenoids

435

1 f o r numbering scheme). Most H e l i a n t h u s s e s q u i t e r p e n e l a c t o n e s a r e c h a r a c t e r i z e d by t h e f u s i o n o f t h e l a c t o n e r i n g t o c a r b o n 6 and t h e presence of a f i v e carbon a n g e l a t e or a n g e l a t e - d e r i v e d a c i d s i d e c h a i n e s t e r i f i e d t o p o s i t i o n 8. Four s t r u c t u r a l t y p e s o f s e s q u i t e r pene l a c t o n e s have been i s o l a t e d f r o m H e l i a n t h u s : germacrolides h e l i a n g o l i d e s , e u d e s m a n o l i d e s and g u a i a n o l i d e s ( F i g u r e 1). The b i o s y n t h e t i c a l l y - s i m p l e r g e r m a c r o l i d e and h e l i a n g o l i d e t y p e s a r e t h e most common i n t h e genus. Four s t r u c t u r a l t y p e s o f d i t e r p e n e s a r e known from H e l i a n t h u s ; l a b d a n e s , k a u r a n e s , a t i s i r a n e s and t r a c h y l o b a n e s ( F i g u r e 2), w i t h t h e t e t r a c y c l i c kaurane t y p e b e i n g t h e most w i d e s p r e a d i n t h e genus. The m a j o r i t y o f H e l i a n t h u s d i t e r p e n e s have an α-oriented c a r b o x y l i c a c i d f u n c t i o n a t t a c h e d t o c a r b o n 4.


f

T o x i c i t y and a n t i f e e d a n t a c t i v i t y l a c t o n e s t o sunflower i n s e c t s

of

Helianthus

sesquiterpene

T e r p e n o i d s i s o l a t e d i n l a r g e q u a n t i t i e s f r o m r e s i s t a n t s p e c i e s were tested with several sunflower insect pests. Since sesquiterpene l a c t o n e s were p r e s e n t i n much h i g h e r c o n c e n t r a t i o n s t h a n d i t e r p e n e s i n t h e most r e s i s t a n t s p e c i e s s t u d i e d , our i n v e s t i g a t i o n s emphasized s e s q u i t e r - p e n e l a c t o n e s . Three s p e c i e s o f i n s e c t s were used i n t h e s e s t u d i e s : Spodoptera e r i d a n i a , M e l a n o p l u s s a n g u i n i p e s and Homoeosoma electellum. S p o d o p t e r a e r i d a n i a ( L e p i d o p t e r a : N o c t u i d a e ) - t h e s o u t h e r n armyworm. T h i s s p e c i e s was chosen f o r s t u d y because i t i s c l o s e l y r e l a t e d t o S. e x i g u a , t h e b e e t armyworm, an i n s e c t w h i c h has p e r i o d i c a l l y damaged f i e l d s o f c u l t i v a t e d s u n f l o w e r i n t h e s o u t h e r n G r e a t P l a i n s (47). A l a b o r a t o r y c o l o n y o f S^ e x i g u a c o u l d n o t be s u c c e s s f u l l y m a i n t a i n e d . Both S^ e r i d a n i a and S^ e x i g u a a r e t e r m e d g e n e r a l i s t f e e d e r s because t h e y c a n f e e d on v a r i o u s o r g a n s o f a v a r i e t y o f t a x o n o m i c a l l y u n r e l a t e d p l a n t s (48, 49). E x p e r i m e n t s on £L e r i d a n i a were p e r f o r m e d i n c o l l a b o r a t i o n w i t h Dr. K. N a k a n i s h i ' s l a b o r a t o r y a t C o l u m b i a University. The g r o w t h o f S^ e r i d a n i a l a r v a e was s i g n i f i c a n t l y r e d u c e d by s e s q u i t e r p e n e l a c t o n e s added t o t h e i r d i e t . Two sesquiterpene l a c t o n e s were used i n t h e s e t e s t s : 8 3 -sarracinoyloxycumambranolide (8 3 SC) f r o m H e l i a n t h u s m a x i m i l i a n i (18) and d e s a c e t y l e u p a s s e r i n f r o m H. m o l l i s (13) ( F i g u r e 3 ) . T h e s e c o m p o u n d s w e r e a d d e d t o an a r t i f i c i a l d i e t a t c o n c e n t r a t i o n s o f 0.1% a n d 1.0% a n d f e d t o f i f t h i n s t a r l a r v a e o f S^ e r i d a n i a . A t a c o n c e n t r a t i o n o f 1.0%, both compounds c a u s e d s i g n i f i c a n t g r o w t h i n h i b i t i o n ( T a b l e 1). Both compounds a r e p r e s e n t i n H e l i a n t h u s l e a v e s a t l e v e l s o f 1-5%. L a r v a e o f S^ e r i d a n i a were a l s o s u b j e c t e d t o p r e f e r e n c e t e s t s t o see i f t h e y would a v o i d i n g e s t i n g s e s q u i t e r p e n e l a c t o n e s i f g i v e n t h e c h o i c e o f f e e d i n g on t r e a t e d o r u n t r e a t e d f o o d . Starved f i f t h i n s t a r l a r v a e were s i m u l t a n e o u s l y p r e s e n t e d w i t h bean l e a v e s ( P h a s e o l u s v u l g a r i s ) c o a t e d w i t h a 1% a c e t o n e s o l u t i o n i n w h i c h a s e s q u i t e r p e n e l a c t o n e had been d i s s o l v e d and bean l e a v e s c o a t e d w i t h solvent only. A f t e r 24 h o u r s , l e a v e s c o a t e d w i t h 8 3 SC s h o w e d s i g n i f i c a n t l y l e s s f e e d i n g than the s o l v e n t - c o a t e d c o n t r o l s , but l a r v a e showed no p r e f e r e n c e between c o n t r o l s and l e a v e s c o a t e d w i t h desacetyleupasserin (Table 2).




Germacrolides

Heliangolides

Guaianolides

F i g u r e 1. S t r u c t u r a l Helianthus.

types o f sesquiterpene

labdane

kaurane

F i g u r e 2.

lactones i n

atisirane

trachylobane

S t r u c t u r a l types o f diterpenes i n Helianthus.


30.

GERSHENZON ET AL.

437


ο


80-Sarracinoyloxycumambranolide (8pSC )

Ο Desacetyleupasserin Figure 3. Sesquiterpene

lactones used i n the insect bioassays.

Table 1. E f f e c t of two Helianthus sesquiterpene the growth of S^ eridania larvae.*

compound and concentration 8 3 SC control 0.1% 1.0% desacetyleupasserin control 0.1% 1.0%

1st

lactones on

average d a i l y weight gain, mg 4th day dav 2nd day 3rd day

122 128 -28** 97 76 44**

97 102 -14** 83 67 56**

143 118 _g** 76 64 45**

82 71 -11** 45 40 50

•Sesquiterpene lactones were added t o a r t i f i c i a l d i e t s at concentra tions indicated. Ten early f i f t h instar larvae were raised on each concentration. Gain i n weight was measured d a i l y f o r each individual. Negative numbers indicate average weight loss f o r larvae i n that group. • • S i g n i f i c a n t l y d i f f e r e n t from control at 1% l e v e l ( t - t e s t ) .



438


Melanoplus sanguinipes (Orthoptera: Acrididae) - the migratory grasshopper. G r a s s h o p p e r s o c c a s i o n a l l y damage c u l t i v a t e d s u n f l o w e r i n t h e U n i t e d S t a t e s (4). M e l a n o p l u s s a n g u i n i p e s i s a g e n e r a l i s t f e e d e r and a major c r o p p e s t s p e c i e s w i d e l y d i s t r i b u t e d i n N o r t h A m e r i c a (50). 8 3 SC a d d e d t o t h e d i e t o f M^_ s a n g u i n i p e s a t a c o n c e n t r a t i o n o f 1% h a d no e f f e c t on t h e g r o w t h o r s u r v i v o r s h i p o f t h i s i n s e c t o r i t s r a t e o f development t o t h e a d u l t s t a g e ( T a b l e 3). However, 8 3 SC was shown t o be a s i g n i f i c a n t f e e d i n g d e t e r r e n t i n p r e f e r e n c e t e s t s w i t h M. s a n g u i n i p e s . G r a s s h o p p e r s t h a t had been s t a r v e d f o r 48 h o u r s were g i v e n t h e c h o i c e o f f e e d i n g on s u c r o s e - t r e a t e d nitrocellulose membrane f i l t e r d i s k s w i t h and w i t h o u t added 83 SC. 8 3 SC was d r i e d o n t o t h e d i s k s i n c h l o r o f o r m s o l u t i o n s a t l e v e l s o f 0.25%, 2.5% a n d 25% o f t h e d r y w e i g h t o f t h e d i s k s . C o n t r o l d i s k s were t r e a t e d w i t h chloroform only. A t a l l l e v e l s o f 8 3SC t e s t e d , sanguinipes consumed more o f t h e c o n t r o l d i s k s t h a n t h e t e s t d i s k s (Table 4). Homoeosoma e l e c t e l l u m ( L e p i d o p t e r a ; P y r a l i d a e ) - t h e s u n f l o w e r moth. I n c o n t r a s t t o S^ e r i d a n i a a n d M^ s a n g u i n i p e s , H. e l e c t e l l u m i s a s p e c i a l i s t w h i c h f e e d s on t h e i n f l o r e s c e n c e s o f a few s p e c i e s o f t h e Asteraceae (4, 5 1 ) . Since s e s q u i t e r p e n e l a c t o n e s a r e found i n s e v e r a l p a r t s o f t h e i n f l o r e s e n c e s o f Helianthus species which are c o n s i d e r e d t o be r e s i s t a n t t o t h e s u n f l o w e r moth (52), i t was t h o u g h t t h a t t h e s e compounds m i g h t s e r v e t o l i m i t t h e damage caused by t h i s insect. L a r v a e o f IL e l e c t e l l u m were r a i s e d on a r t i f i c i a l wheat germbased d i e t s t o w h i c h v a r y i n g c o n c e n t r a t i o n s o f 8 3 SC had been added. T h i s compound was d i s s o l v e d i n acetone, c o a t e d on c e l l u l o s e powder under vacuum and t h e n mixed i n t o t h e d i e t a t c o n c e n t r a t i o n s o f 0.01%, 0.1% and 1%. The c o n t r o l d i e t c o n t a i n e d c e l l u l o s e powder w h i c h had been soaked i n a c e t o n e and d r i e d under vacuum. A t l e v e l s o f 0.1% and 1%, 8 3 SC s i g n i f i c a n t l y r e d u c e d p u p a l w e i g h t ( T a b l e 5 ) . H o w e v e r , l a r v a l s u r v i v a l and development t i m e were n o t a f f e c t e d . F o r f e e d i n g p r e f e r e n c e t e s t s , 8 3 SC was a d d e d t o s q u a r e s o f a r t i f i c i a l d i e t a t a c o n c e n t r a t i o n o f 5%. S t a r v e d H^_ e l e c t e l l u m l a r v a e were p l a c e d i n t h e c e n t e r o f a d i s h c o n t a i n i n g both t r e a t e d and c o n t r o l d i e t s q u a r e s a n d a l l o w e d t o f e e d . A f t e r two h o u r s , s i g n i f i c a n t l y more f i r s t and second i n s t a r l a r v a e were found f e e d i n g on t h e c o n t r o l d i e t (73%) t h a n on t h e t r e a t e d d i e t (12%) ( T a b l e 6 ) . However, t h e t h i r d , f o u r t h a n d f i f t h i n s t a r s d i d n o t show a s i g n i f i c a n t preference i n t h i s experiment. The r e s u l t s o f t h e s e b i o a s s a y s i n d i c a t e t h a t s e s q u i t e r p e n e lactones i s o l a t e d from species of Helianthus r e s i s t a n t t o major insect pests of c u l t i v a t e d sunflower a r e t o x i n s and f e e d i n g d e t e r r e n t s t o some o f t h e s e i n s e c t s . P r e v i o u s i n v e s t i g a t i o n s have shown t h a t s e s q u i t e r p e n e l a c t o n e s have t o x i c and a n t i f e e d a n t a c t i v i t i e s t o w a r d s a v a r i e t y o f phytophagous i n s e c t s (53-62). E v o l u t i o n a r i l y , s e s q u i t e r p e n e l a c t o n e s may have come t o f u n c t i o n as f e e d i n g d e t e r r e n t s because i n s e c t s have been s e l e c t e d f o r t h e i r a b i l i t y t o r e c o g n i z e t h e p r e s e n c e o f t h e s e t o x i c compounds i n p o t e n t i a l f o o d s t u f f s and a v o i d i n g e s t i n g them. Sesquiterpene l a c t o n e s e x h i b i t a number o f t o x i c e f f e c t s a t t h e c e l l u l a r l e v e l , i n c l u d i n g t h e i n h i b i t i o n o f p r o t e i n s y n t h e s i s (63, 64), n u c l e i c a c i d s y n t h e s i s (63-66) and r e s p i r a t o r y enzyme a c t i v i t y (67). The t o x i c i t y of t h e s e compounds i s t h o u g h t t o be due t o t h e i r a b i l i t y t o a l k y l a t e n u c l e o p h i l i c s i t e s on p r o t e i n s (63, 68-70) o r DNA (64, 6 6 ) .


30.

GERSHENZON ET AL.

T a b l e 2.

F e e d i n g d e t e r r e n c e o f two H e l i a n t h u s l a c t o n e s t o S. e r i d a n i a l a r v a e . *

compound


439


8 3 SC desacetyleupasserin

sesquiterpene

average amount o f l e a v e s e a t e n treated control 21.0%** 54.2% 36.7% 30.0%

*1% Acetone s o l u t i o n s o f each s e s q u i t e r p e n e l a c t o n e were c o a t e d on bean l e a v e s . C o n t r o l l e a v e s were c o a t e d w i t h s o l v e n t o n l y . S t a r v e d f i f t h i n s t a r l a r v a e were g i v e n a c h o i c e o f f e e d i n g on c o n t r o l o r t r e a t e d l e a v e s . T e s t s o f each compound a g a i n s t a c o n t r o l were r e p e a t e d w i t h t e n l a r v a 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 a t 5% l e v e l ( t - t e s t f o r p a i r e d comparisons).

T a b l e 3.

E f f e c t o f 8 3SC on t h e growth and development o f Melanoplus s a n g u i n i p e s . *

diet control average a d u l t w e i g h t , g ± s . d . female male average t i m e o f development from f i r s t i n s t a r t o a d u l t , days + s.d.

control with added 8 β SC

.021 0.224 0.187 + .033

0.210 t .015 0.198 ± .024

40.3 f 1.9

41.7 -t 3.6

*Diet consisted of finely-ground freeze-dried rye grass. 8 3SC i n d i c h l o r o m e t h a n e s o l u t i o n was c o a t e d on t h e d i e t a t a l e v e l o f 1% o f t h e d r y w e i g h t o f t h e d i e t and t h e s o l v e n t a l l o w e d t o evaporate. C o n t r o l d i e t was t r e a t e d w i t h s o l v e n t o n l y . Twenty i n s e c t s were i n d i v i d u a l l y r e a r e d on each t r e a t m e n t . T h e r e were no s i g n i f i c a n t d i f f e r e n c e s between t h e t r e a t m e n t s .



440

Table

4.

E f f e c t o f 8 ÉISC sanguinipes·


p e r c e n t a g e o f 8 (3SC per d r y wgt. o f d i s k 0.25 2.5 25

on

the

feeding

preference

of

a v e r a g e amount o f c o n t r o l d i s k s5 e a t e n i n e x c e s s o f t e s t d i s k s , cit£ ± s.d. . 0.30 + 0.46** 1.19 + 0.99** 0.97 + 1.25**

•Grasshoppers t h a t had been s t a r v e d f o r 48 h o u r s were p r e s e n t e d w i t h 5 cm n i t r o c e l l u l o s e membrane f i l t e r d i s k s t o w h i c h 5% s u c r o s e s o l u t i o n had been added. T e s t d i s k s were t r e a t e d w i t h 8 3 SC i n chloroform solution while control disks received chloroform only. A l l d i s k s were a i r - d r i e d . A r e a o f d i s k e a t e n was deter mined w i t h an a r e a meter. * * S i g n i f i c a n t p r e f e r e n c e a t t h e 1% l e v e l ( t - t e s t f o r p a i r e d c o m p a r i sons ) ·

T a b l e 5.

E f f e c t o f 8 3 SC on t h e s u r v i v a l , development and weight of electellum larvae.*

survival to adult, % development t i m e (from h a t c h t o a d u l t e c l o s i o n ) , days pupal weight, g

control 82 25.7 34.4

concentration i n d i e t 0.01% 0,1% 89 83 25.6 33.3

25.7 32.4**

pupal

1.0% 77 25.7 31.3**

*8 3 SC was d i s s o l v e d i n a c e t o n e and c o a t e d on c e l l u l o s e powder w h i c h was added t o t h e d i e t t o g i v e t h e c o n c e n t r a t i o n s i n d i c a t e d . C o n t r o l d i e t c o n t a i n e d c e l l u l o s e powder w h i c h had been soaked i n acetone. Each c o n c e n t r a t i o n was t e s t e d on a t l e a s t 100 l a r v a e . * * 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 s a t t h e 5% l e v e l ( a n a l y s i s o f v a r i a n c e ) . Data on development t i m e and s u r v i v a l d i d not show any s i g n i f i c a n t e f f e c t o f 8 3 SC dose. Development t i m e d a t a were s u b j e c t e d t o an a n a l y s i s o f v a r i a n c e and s u r v i v a l d a t a t o a t e s t o f independence u s i n g t h e G - s t a t i s t i c .


30. GERSHENZON ET AL.


Possible role of s u n f l o w e r moth.

Insect Antifeedant Terpenoids sesquiterpene

lactones

i n resistance

441 to the

Homoeosoma e l e c t e l l u m , t h e s u n f l o w e r m o t h , i s c u r r e n t l y t h e m o s t s e r i o u s p e s t o f c u l t i v a t e d s u n f l o w e r i n t h e u n i t e d S t a t e s (4). I n t h e p r e v i o u s s e c t i o n , we s h o w e d t h a t s e s q u i t e r p e n e l a c t o n e s f r o m s p e c i e s o f H e l i a n t h u s r e s i s t a n t t o FL e l e c t e l l u m a r e t o x i c a n d a n t i f e e d a n t t o IL e l e c t e l l u m l a r v a e when added t o a r t i f i c i a l d i e t s . To e v a l u a t e t h e p o s s i b l e i n v o l v e m e n t o f s e s q u i t e r p e n e l a c t o n e s i n r e s i s t a n c e t o H. e l e c t e l l u m i n t h e i n t a c t p l a n t , i t i s n e c e s s a r y t o consider the feeding habits of this insect i n r e l a t i o n to the l o c a t i o n of sesquiterpene lactones i n the i n f l o r e s e n c e s of r e s i s t a n t species of Helianthus. S e s q u i t e r p e n e l a c t o n e s a r e found i n g l a n d u l a r t r i c h o m e s on t h e t e r m i n a l a n t h e r appendages, i m m e d i a t e l y a d j a c e n t t o t h e p o l l e n (52) ( F i g u r e 4 ) . Y o u n g H. e l e c t e l l u m l a r v a e ( f i r s t a n d second i n s t a r s ) e a t p r i n c i p a l l y p o l l e n , w h i l e l a t e r i n s t a r s f e e d on a variety of f l o r a l parts: corollas, styles, ovaries, developing achenes and p a r t s o f t h e r e c e p t a c l e (4, 51, 7 1 ) . How m i g h t young IL e l e c t e l l u m l a r v a e o b t a i n p o l l e n ? Before the f l o r e t s open, t h e p o l l e n i s p r o d u c e d and s t o r e d i n t h e a n t h e r s . To r e a c h t h e p o l l e n i n an unopened f l o r e t , l a r v a e must c r a w l o r e a t t h e i r way i n t o t h e f l o r e t a n d t h e n e a t i n t o t h e a n t h e r s . The s e s q u i t e r p e n e l a c t o n e - c o n t a i n i n g g l a n d u l a r t r i c h o m e s on t h e a n t h e r t i p s seem t o be s i t u a t e d i n j u s t t h e r i g h t l o c a t i o n t o p r e v e n t t h e l a r v a e from r e a c h i n g t h e p o l l e n ( F i g u r e 4 ) . A f t e r a f l o r e t opens, p o l l e n i s shed i n t o t h e c e n t e r o f a c y l i n d e r f o r m e d by t h e f u s e d a n t h e r s and t h e s t y l e b r a n c h pushes up t h r o u g h t h e c y l i n d e r r a i s i n g t h e p o l l e n above t h e a n t h e r s f o r p r e s e n t a t i o n t o p o t e n t i a l p o l l i n a t o r s (72). To r e a c h t h e p o l l e n a t t h i s s t a g e , young l a r v a e would have t o c r a w l up and o v e r t h e a n t h e r s ( F i g u r e 4). The s e s q u i t e r p e n e l a c t o n e - c o n t a i n i n g g l a n d u l a r t r i c h o m e s on t h e a n t h e r appendages c o u l d a l s o d e t e r l a r v a e f r o m t h e a n t h e r s ( o r s t i g m a s ) i n t h i s manner a f t e r t h e f l o r e t s a r e open. However, t h e r e i s as y e t no e v i d e n c e t o s u p p o r t t h i s p o s s i b i l i t y . The p r o x i m i t y o f s e s q u i t e r p e n e lactones to the pollen i n r e s i s t a n t species of Helianthus, i n conjunction with the deleterious e f f e c t s o f t h e s e compounds on IL e l e c t e l l u m when i n c o r p o r a t e d i n t o artificial diets, suggests that the presence of sesquiterpene l a c t o n e s may h a v e b e e n s e l e c t e d f o r b e c a u s e t h e s e c o m p o u n d s c a n r e d u c e s u n f l o w e r moth prédation by p r e v e n t i n g t h e young l a r v a e f r o m r e a c h i n g t h e i r p r i n c i p a l food source. I f young IL e l e c t e l l u m l a r v a e c a n n o t o b t a i n s u f f i c i e n t p o l l e n , t h e y may n o t s u r v i v e t o c a u s e f u r t h e r damage t o t h e i n f l o r e s e n c e . O b s e r v a t i o n s on t h e c u l t i v a t e d l i n e s of sunflower support the r o l e of sesquiterpene lactones i n r e s i s t a n c e t o t h e s u n f l o w e r moth. C u l t i v a r s w h i c h a r e f r e q u e n t l y h e a v i l y damaged by IL e l e c t e l l u m l a r v a e were found t o have much l o w e r d e n s i t i e s o f g l a n d u l a r t r i c h o m e s on t h e i r a n t h e r s t h a n t h e r e s i s t a n t s p e c i e s o f H e l i a n t h u s (52). T h e r e f o r e , i t may be p o s s i b l e t o i n c r e a s e t h e r e s i s t a n c e o f c u l t i v a t e d s u n f l o w e r t o IL e l e c t e l l u m b y b r e e d i n g f o r i n c r e a s e d concentrations of sesquiterpene lactone-containing glandular t r i c h o m e s on t h e a n t h e r t i p s . T h e d e v e l o p m e n t o f new c u l t i v a t e d l i n e s w i t h h i g h d e n s i t i e s o f g l a n d u l a r t r i c h o m e s may p r o v e t o be an



442

Table

6.

larval

E f f e c t o f 8 |3SC on t h e f e e d i n g p r e f e r e n c e larvae.*

instar


1 s t and 2nd 3 r d , 4 t h and 5 t h

feeding on c o n t r o l 73%** 45%

o f IL e l e c t e l l u m

not feeding 15% 23%

f e e d i n g on treated diet 12% 32%

* L a r v a e were a l l o w e d t o choose between f e e d i n g on a d i e t c o n t a i n i n g 5% 8 BSC and a c o n t r o l d i e t . Twenty l a r v a e o f each i n s t a r were tested. R e s u l t s f o r f i r s t and second i n s t a r s , on t h e one hand, and f o r t h i r d , f o u r t h and f i f t h i n s t a r s , on t h e o t h e r , were s i m i l a r and t h e r e f o r e combined. * * S i g n i f i c a n t p r e f e r e n c e a t t h e 5% l e v e l ( c h i - s q u a r e t e s t ) .

Unopened disk floret (cut-away view )

Disk floret (pollen-donor phase)

F i g u r e 4. L o c a t i o n o f s e s q u i t e r p e n e l a c t o n e - c o n t a i n i n g t r i c h o m e s on s u n f l o w e r d i s k f l o r e t s .

glandular



30.

GERSHENZON ET AL.


443

e f f i c i e n t and p r a c t i c a l way o f c o n t r o l l i n g t h e s u n f l o w e r moth a t an e a r l y s t a g e i n i t s development. Glandular trichomes a c t t o put the d e t e r r e n t c o m p o u n d s when a n d w h e r e t h e y a r e n e e d e d f o r d e f e n s i v e p u r p o s e s w i t h o u t t h e h a z a r d s and expense o f a p p l y i n g s y n t h e t i c insecticides. G l a n d u l a r t r i c h o m e c o n s t i t u e n t s have been a s s o c i a t e d w i t h i n s e c t r e s i s t a n c e i n a number o f o t h e r c r o p s , i n c l u d i n g c o t t o n , tomato, p o t a t o , t o b a c c o and a l f a l f a (73). However, t h i s i s t h e f i r s t t i m e t h a t g l a n d u l a r t r i c h o m e s o r any p l a n t n a t u r a l p r o d u c t has been i m p l i c a t e d i n t h e d e f e n s e o f p o l l e n a g a i n s t prédation. S e s q u i t e r p e n e l a c t o n e - c o n t a i n i n g g l a n d u l a r t r i c h o m e s may n o t s e r v e as e f f e c t i v e d e f e n s e s a g a i n s t o l d e r IL e l e c t e l l u m l a r v a e s i n c e , i n o u r p r e f e r e n c e b i o a s s a y s , l a t e r i n s t a r s ( t h i r d , f o u r t h and f i f t h i n s t a r s ) w e r e n o t s i g n i f i c a n t l y d e t e r r e d b y 8 β SC ( T a b l e 6 ) . I n a d d i t i o n , o l d e r l a r v a e f e e d i n g on c u l t i v a r s h a v e b e e n o b s e r v e d t o chew t h r o u g h t h e b a s e o f t h e c o r o l l a s o f u n o p e n e d f l o r e t s t o r e a c h t h e p o l l e n (71). T h i s b e h a v i o r a l l o w s t h e l a r v a e t o a v o i d any g l a n d u l a r t r i c h o m e s w h i c h m i g h t be p r e s e n t on t h e a n t h e r t i p s . Other c h e m i c a l a n d m o r p h o l o g i c a l f e a t u r e s m i g h t be i m p o r t a n t i n t h e r e s i s t a n c e t o o l d e r H. e l e c t e l l u m l a r v a e , s u c h a s t h e p r e s e n c e o f a s o - c a l l e d "phytomelanin" l a y e r i n t h e w a l l s o f d e v e l o p i n g achenes w h i c h may f o r m a b a r r i e r t o l a r v a l f e e d i n g (74). T h e p r o t e c t i o n o f s u n f l o w e r f r o m damage by a l l s t a g e s o f IL e l e c t e l l u m l a r v a e may t h u s r e q u i r e t h e development o f c u l t i v a r s w i t h a c o m b i n a t i o n o f d e f e n s i v e traits. Acknowledgments We t h a n k V. L e s k i n e n a n d K. N a k a n i s h i f o r t h e S. e r i d a n i a t e s t d a t a , G. G r a d o w i t z f o r t e c h n i c a l a s s i s t a n c e w i t h t h e NL s a n g u i n i p e s s t u d i e s a n d M. C o n o l e y f o r h e l p w i t h t h e H^ e l e c t e l l u m e x p e r i m e n t s . This w o r k was s u p p o r t e d by g r a n t s t o T. J . M a b r y f r o m t h e N a t i o n a l I n s t i t u t e s o f H e a l t h (HDO-4488) a n d t h e R o b e r t A. W e l c h F o u n d a t i o n (F-130) and a g r a n t t o J . Gershenzon f r o m t h e S u n f l o w e r A s s o c i a t i o n of A m e r i c a . T h i s i s c o n t r i b u t i o n No. 84-13-A f r o m t h e Department o f Entomology, Kansas A g r i c u l t u r a l E x p e r i m e n t S t a t i o n . We acknowledge t h e use o f t h e U n i v e r s i t y o f Texas B r a c k e n r i d g e F i e l d L a b o r a t o r y .

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RECEIVED November 23, 1984


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