Ascorbic Acid - American Chemical Society

13 Ascorbic A c i d and the Growth and Development of Insects

Downloaded by IOWA STATE UNIV on February 21, 2017 | http://pubs.acs.org Publication Date: June 1, 1982 | doi: 10.1021/ba-1982-0200.ch013

KARL J. KRAMER U.S. Grain Marketing Research Laboratory, Agricultural Research, Science and Education Administration, U.S. Department of Agriculture, Manhattan, KS 66502 and Department of Biochemistry, Kansas State University, Manhattan, KS 66506 PAUL A. SEIB Department of Grain Science, Kansas State University, Manhattan, KS 66506

The structural requirements for vitamin C activity in insects were comparable to those observed in guinea pigs. A dietary level of 0.5 mM L-ascorbic acid was necessary for normal development of the tobacco hornworm (Manduca sexta); magnesium 2-O-phosphono-L-ascorbate, so dium 6-O-myristoyl-L-ascorbate, and L-dehydroascorbic acid were equally potent. D-Ascorbic acid, 6-bromo-6-deoxy-L -ascorbic acid, and D-isoascorbic acid were approximately one-half, one-fifth, and one-tenth as effective, respectively. Tissues from M . sexta lacked L-gulono-γ-lactone oxidase, the biosynthetic enzyme usually absent from ascorbate -dependent species. Vitamin C was found in eggs, larval labial gland, hemolymph, gut, muscle, cuticle, adult nervous tissue, and gonads at concentrations ranging from < 10-170 mg/100 g of wet tissue. No ascorbate was detected in larval fat body, Malpighian tubules, or adult salivary gland. In sects reared on an L-ascorbate-deficient diet contained no detectable L-ascorbic acid. Some possible physiological actions of the vitamin in insects are discussed.

data are available on the insect's requirement for LConsiderable ascorbic acid. Dietary vitamin C is needed for normal growth, molting, and fertility of many insects, and vitamin C, or another com pound with similar biological properties, is probably an essential growth 0065-2393/82/0200-0275$06.00/ 0 © 1982 American Chemical Society Seib and Tolbert; Ascorbic Acid: Chemistry, Metabolism, and Uses Advances in Chemistry; American Chemical Society: Washington, DC, 1982.

276

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ACID

f a c t o r f o r this class of a n i m a l s . A l t h o u g h m o s t insects s u b s i s t i n g o n g r e e n p l a n t s n e e d L - a s c o r b a t e t o d e v e l o p f u l l y (1-6),

i t w a s p r o p o s e d t h a t some

species m a y d i s p e n s e w i t h t h e v i t a m i n o r m a y synthesize i t e i t h e r d e n o v o or r e l y on s y m b i o t i c o r g a n i s m s (7-10).

H o w e v e r , t h e a b i l i t y of c e r t a i n

insects ( o r t h e i r s y m b i o n t s ) to synthesize a s c o r b i c a c i d has n o t b e e n a d e q u a t e l y d e m o n s t r a t e d . T h i s c h a p t e r r e v i e w s some of t h e p r e v i o u s w o r k on t h e r o l e of a s c o r b i c a c i d in insects a n d i n c l u d e s results of efforts to d e v e l o p a bioassay f o r v i t a m i n C u s i n g a n insect, m e a s u r e t h e g r o w t h p r o m o t i n g a c t i v i t y of c o m p o u n d s s t r u c t u r a l l y r e l a t e d to L - a s c o r b i c a c i d , d e t e r m i n e d i e t a n d tissue levels of L - a s c o r b a t e i n insects, a n d a s c e r t a i n


w h e t h e r specific tissues in insects are c a p a b l e of c o n v e r t i n g a p u t a t i v e precursor, L-gulono-y-lactone, into vitamin C.

Experimental Animals. Manduca sexta larvae were reared on an agar-based diet (11) at 28 °C and 60% relative humidity with a 16-h photophase. The Indian meal moth, Plodia interpunctella Hubner, and American cockroach, Periplaneta americana L . , were taken from laboratory cultures. Dissection was performed under anesthesia by cooling to 5°C (12). Biological Assay. Prior to bioassay, the hot diet was cooled to 60° C, L-ascorbic acid or a related compound was added, and the mixture was thor oughly blended. Labile derivatives were applied to the surface of the gelled diet at room temperature. Neonate larvae were used in all tests and the growth of larvae on the control diet was compared with that of larvae on test diets. At 1-4-d intervals, up to 40 d, the mean weight of ten to twenty animals was determined. Fecal matter was removed at each observation. Test compounds were obtained or prepared as described previously (5). Paper Chromatography. One-tenth of a gram of tissue was homogenized in 0.25 m L of 2% (w/v) metaphosphoric acid at 4°C. Descending paper chromatography was done on Whatman #1 paper using ethyl acetate:acetic acid: water (6:3:2) as developing solvent. Ascorbic acid was detected by dipping the chromatogram sequentially in 0.10 m L of saturated silver nitrate mixed with 20 m L of acetone containing 0.1 m L of concentrated ammonium hydroxide, 1 M N a O H in 95% ethanol, 0.2 M aqueous sodium thiosulfate, and water (13). The detection limit was 2 /xg after chromatography. High Performance Liquid Chromatography. Tissue extracts were ana lyzed with a Varian model 5020 liquid chromatograph equipped with a Rheodyne model 7120 loop injector valve, a Tracor 970 variable wavelength detector set at 257 nm, an automated Hewlett-Packard 3385A printer-plotter system for determining retention times and peak areas, and a Waters n Bondapak column (3.9 mm i.d. X 300 mm) for carbohydrate analysis. The buffer was eluted isocratically at 1 m L / m i n with a 1:4 (v/v) mixture of 0.01 M monobasic sodium phosphate (pH 4.46) and methanol. The minimum amount detectable was 10 ng. L-Gulonolactone Oxidase Assay. Tissues were assayed for L-gulonolactone oxidase by the method of Azaz et al. (14). Weighed portions of tissue (50-200 mg) were homogenized in 2 m L of 50 m M sodium phosphate ( p H 7.4) containing 0.2% sodium deoxycholate. Homogenates were centrifuged at

Seib and Tolbert; Ascorbic Acid: Chemistry, Metabolism, and Uses Advances in Chemistry; American Chemical Society: Washington, DC, 1982.

13.

Ascorbic Acid ir Insect Development

K R A M E R AND SEIB

277

5000 g for 10 m i n at 4°C, and 1-mL aliquots of the supernatant were incubated w i t h 2 m M L-gulono-y-lactone (Sigma C h e m i c a l Company) for 60 m i n at 35°C. Ascorbate was measured by the 2,4-dinitrophenylhydrazine method of Roe and Kuether (15) as modified by Geshwind et al. ( 1 6 ) . C h i c k e n kidney was assayed as a control tissue rich i n L-gulonolactone oxidase. Results Bioassay.

T h e effect of L - a s c o r b i c a c i d o n t h e d e v e l o p m e n t rate of

M . sexta is s h o w n i n F i g u r e 1 ( 5 ) .

A l l neonate l a r v a e d e v e l o p e d

into

adults o n t h e a r t i f i c i a l d i e t that c o n t a i n e d 0.5 m M L - a s c o r b i c a c i d . A l s o ,


larvae raised on that diet exhibited a n o r m a l growth curve, a n d were r o b u s t a n d b r i g h t b l u e - g r e e n i n color. 35 d w i t h l a r v a l - p u p a l ecdysis

N o r m a l development occurred i n

o c c u r r i n g at d a y

15 a n d p u p a l - a d u l t

ecdysis at d a y 35. H i g h e r levels of L - a s c o r b i c a c i d w e r e not m o r e effec t i v e , b u t l o w e r ones w e r e i n a d e q u a t e for the h o r n w o r m . A s the a m o u n t of L - a s c o r b i c a c i d w a s decreased i n the diet, p a t h o l o g i c a l effects a p p e a r e d after a f e e d i n g

period that depended

on the v i t a m i n concentration.

A n i m a l s r e a r e d o n a n ascorbate-deficient d i e t w e r e r e d u c e d i n size a n d colored a dull yellowish-green.

A b n o r m a l i t i e s i n c u t i c l e soon b e c a m e

a p p a r e n t . E x t r e m i t i e s s u c h as m o u t h parts a n d tarsi e x h i b i t e d p r e m a t u r e d a r k e n i n g of c u t i c l e .

N a v o n (6)

o b s e r v e d s i m i l a r effects i n t h e c o t t o n

l e a f w o r m , Spodoptera littoralis. I n a l l tests, l a r v a e a p p e a r e d n o r m a l to the s e c o n d instar, p r o b a b l y because of a n a m o u n t of L - a s c o r b i c a c i d d e r i v e d f r o m p a r e n t insects. I n l a r v a e o n diets l a c k i n g i n L - a s c o r b i c a c i d , p a t h o l o g i c a l consequences o c c u r r e d at the t h i r d instar.

A t the b e g i n n i n g of

t h e t h i r d m o l t i n g p e r i o d , t h e insects b e g a n to s h r i v e l a n d 1 d later b e c a m e m o r i b u n d . L a r v a e f e d m e d i u m c o n t a i n i n g 0.05 m M L - a s c o r b i c a c i d w e r e s i m i l a r l y affected, b u t at o n e s t a d i u m later. F i f t y p e r c e n t of t h e l a r v a e f e d d i e t s u p p l e m e n t e d w i t h 0.25 m M v i t a m i n C d i e d i n the p r e p u p a l stage; t h e other h a l f u n d e r w e n t p u p a l a n d a d u l t eclosion 3 - 6 d later t h a n the control group. W e h a v e u s e d the g r o w t h effects a n d pathologies associated

with

L - a s c o r b i c a c i d deficiency as a basis for the d e t e r m i n a t i o n of the b i o l o g i c a l p o t e n c y of r e l a t e d c o m p o u n d s

(Table I ) . A t a dietary concentra

t i o n of 0.5 m M , L - a s c o r b i c a c i d a n d d e h y d r o a s c o r b i c

acid were

fully

a c t i v e , as w e l l as some ester d e r i v a t i v e s i n c l u d i n g the 6-myristate a n d 2-phosphate compounds.

T h e insect m a y be m e t a b o l i c a l l y l i k e the g u i n e a

p i g b e c a u s e b o t h w e r e able to u t i l i z e those esters (17).

Carboxylesterases

a n d phosphatases p r o b a b l y c o n v e r t e d those d e r i v a t i v e s to the free v i t a m i n (18).

T h e 6 - b r o m o c o m p o u n d w a s less a c t i v e a n d a p p a r e n t l y c a n n o t b e

m e t a b o l i z e d to L - a s c o r b i c a c i d or o n l y p o o r l y so. O n e of the least a c t i v e c o m p o u n d s 2-sulfate ester of L - a s c o r b i c a c i d .

i n the insect bioassay w a s t h e

T o d e v e l o p n o r m a l l y the h o r n w o r m



4

8

12

20

24

28

TIME OF DEVELOPMENT (DAYS)

16

32

36

40

Growth curves of M. sexta fed diet containing added 0.50 mM (-*-), 0.25 mM ( - • - ) , 0.05 , or 0.00 mM (-0-) vitamin C. Open arrow and closed arrow denote the time of pupal and adult ecdysis, respectively (5).

0


13.

Table I.

Effect of L-Ascorbic A c i d and Related Compounds on G r o w t h of M . sexta and Cavia cobaya Relative Hornworm

Compound


279

Ascorbic Acid ir Insect Development

KRAMER AND SEIB

L-£/ireo-Hex-2-enonic a c i d y - l a c t o n e (L-ascorbic acid) Sodium 6-0-myristoyl-L-ascorbate Magnesium 2-0-phosphono-L-ascorbate D-£/ireo-Hex-2-enonic a c i d y - l a c t o n e (D-ascorbic acid) 6-Eromo-6-deoxy-L-ascorbic acid D-en/£/iro-Hex-2-enonic a c i d y - l a c t o n e (D-isoascorbic acid) Potassium 2-O-sulfo-L-ascorbate L-en/£/iro-Hex-2-enonic a c i d y - l a c t o n e ( L - i s o a s c o r b i c acid) L-£/ireo-Hex-2,3-diulosic a c i d y - l a c t o n e (L-dehydroascorbic acid)

Activity

0

Guinea

100 100 100

100 100 100 40 ± 20 ±

10 10

10 ±

Pig

0 not a v a i l a b l e

5

10

5 0

0

0

100

100

" I n s e c t growth a c t i v i t y is expressed as the a m o u n t of c o m p o u n d relative to L-ascorbic acid (0.50 m M ) required for > 8 0 % of the test a n i m a l s to a t t a i n a weight of 1 g i n 10 d (5).

r e q u i r e d a t w e n t y times greater c o n c e n t r a t i o n of this conjugate ( 1 0 m M ) . A p p a r e n t l y , M . sexta does not m e t a b o l i z e the sulfate ester b a c k to L a s c o r b i c a c i d because i t p r o b a b l y l a c k s a s u l f o h y d r o l a s e e n z y m e .

Pre

l i m i n a r y results i n d i c a t e d t h a t t h e 2-sulfate d e r i v a t i v e w a s a b o u t h a l f as a c t i v e as L - a s c o r b i c a c i d i n the s o u t h w e s t e r n c o r n b o r e r , Diatraea grandiosella D y a r mM

(19).

T h a t species r e q u i r e d a d i e t a r y s u p p l e m e n t of

21

L - a s c o r b i c a c i d for o p t i m a l g r o w t h ( 2 0 ) , a p p r o x i m a t e l y f o r t y t i m e s

h i g h e r t h a n the l e v e l r e q u i r e d b y M . sexta. T h e s e differences m a y express the

m e t a b o l i c needs of i n d i v i d u a l species.

the

tobacco h o r n w o r m ( T a b l e I ) .

C5

affected a c t i v i t y a n d i n d i c a t e d t h a t the g e o m e t r y of C 5 w a s

T h r e e stereoisomers of L - a s c o r b i c a c i d w e r e also b i o a s s a y e d u s i n g C o n f i g u r a t i o n a l changes at C 4 a n d more

c r i t i c a l for a c t i v i t y t h a n t h a t of C 4 . T h e e n a n t i o m e r , D-ascorbic a c i d , h a d approximately 4 0 % had

1 0 % activity.

a c t i v i t y , w h i l e t h e C 5 e p i m e r , D-isoascorbic a c i d , T h e r e l a t i v e p o t e n c y of those isomers is r e v e r s e d i n

v e r t e b r a t e a n d i n v e r t e b r a t e a n i m a l s . W i t h D-isoascorbic, 2 - 1 0 %

activity

i n o t h e r insects w a s r e p o r t e d (20-22), b u t this c o m p o u n d d i d not p r o m o t e d e v e l o p m e n t of t h e c o t t o n leaf w o r m (23).

L - I s o a s c o r b i c a c i d h a d no

a c t i v i t y i n the h o r n w o r m or g u i n e a p i g . L-Dehydroascorbic acid, a derivative w i t h potent vitamin activity i n vertebrates, w a s i n a c t i v e i n o u r bioassay w h e n i t w a s m i x e d w i t h h o t d i e t p r i o r to g e l a t i o n .

However, when we

applying dehydroascorbic

r e p e a t e d t h e bioassay

a c i d to t h e surface of

by

the g e l l e d d i e t , t h e


280

ASCORBIC

ACID

o x i d i z e d f o r m p r o v e d to b e as effective as L - a s c o r b i c a c i d i n p r o m o t i n g insect g r o w t h .

Apparently, dehydroascorbic

acid w a s destroyed at the

e l e v a t e d t e m p e r a t u r e {24). T h e p o s s i b i l i t y that L - a s c o r b i c a c i d w a s e x e r t i n g its g r o w t h - p r o m o t i n g effect o n t h e h o r n w o r m as a nonspecific r e d u c i n g agent w a s tested. O r g a n i c a n d i n o r g a n i c agents quinone,

pyrocatechol,

s u c h as reductones, t o c o p h e r o l ,

thiols, ferrous

sulfate, a n d s o d i u m

hydro-

dithionite

e x h i b i t e d n o a c t i v i t y . T h e c a r b o n r i n g a n a l o g , r e d u c t i c a c i d , w a s also inactive.

O b v i o u s l y , t h e t o b a c c o h o r n w o r m d i s p l a y e d stereoselectivity

for L - a s c o r b i c a c i d a n d is a g o o d m o d e l f o r t h e s t u d y o f s t r u c t u r e -


activity relationships. Ascorbate Levels i n Tissues.

S e v e r a l tissues w e r e d i s s e c t e d

M . sexta a n d a n a l y z e d f o r L - a s c o r b i c a c i d b y h i g h p e r f o r m a n c e chromatography

(HPLC)

( F i g u r e 2 ) , paper

chromatography,

from liquid

or the

dinitrophenylhydrazine method ( 5 ) . A s anticipated, L-ascorbic a c i d was

0

5

10 0

5

10 0

5

10

RETENTION TIME, MIN. Figure 2. HPLC of L-ascorbic acid from insect tissues (65). A, L-ascorbic acid, 1.7 fxg; B, M . sexta hemolymph (0.01 mL) extract; C, M . sexta labial gland extract, 1.4 mg wet weight.


13.

Ascorbic Acid b- Insect Development

KRAMER AND SEIB

281

present i n n e a r l y a l l tissues ( T a b l e I I ) , a l t h o u g h i t w a s m o s t a b u n d a n t in

larval

labial

gland

and hemolymph,

ranging from

1 to 10

mM.

L - A s c o r b i c a c i d w a s also present at v a r y i n g levels i n eggs, l a r v a l gut, m u s c l e , c u t i c l e , a d u l t nervous tissue, a n d gonads.

F o r comparison, L -

ascorbic a c i d w a s assayed i n d i e t a n d f e c a l m a t t e r at 24 a n d 5

mg/100

g, r e s p e c t i v e l y .

of

the

v i t a m i n w a s a b s o r b e d a n d / o r m e t a b o l i z e d b y tissues. N o ascorbate

was

T h i s result i n d i c a t e d that a p p r o x i m a t e l y 8 0 %

d e t e c t e d i n l a r v a l fat b o d y , M a l p h i g i a n t u b u l e , or a d u l t s a l i v a r y g l a n d . T h u s , insects a p p e a r to be different f r o m vertebrates, w h e r e the highest levels of L - a s c o r b i c a c i d o c c u r i n t h e adrenals a n d nervous tissue

(25).


L - A s c o r b i c a c i d w a s also a n a l y z e d i n tissues f r o m h o r n w o r m s f e d a v i t a m i n - d e f i c i e n t diet. W i t h o u t L - a s c o r b i c a c i d neonate l a r v a e g r e w into the t h i r d instar, b u t d i e d b e f o r e the next m o l t .

These larvae retained

l i t t l e or no v i t a m i n i n tissues ( T a b l e I I ) . A s i m i l a r result w a s c h a r a c t e r istic of fifth instar l a r v a e r e a r e d o n a n ascorbate-deficient d i e t b e g i n n i n g at t h e m i d f o u r t h instar.

These

l a r v a e f a i l e d to

complete

pupation.

A p p a r e n t l y , the diet was t h e sole source of L - a s c o r b i c a c i d a n d w h e n tissues b e c a m e d e p l e t e d , m a j o r p a t h o l o g i c a l consequences ensued.

Table II.

L-Ascorbic A c i d Content of Tissues from M . sexta

Tissue L a b i a l gland Hemolymph B r a i n a n d nerve c o r d Gonad Egg Gut Muscle Cuticle M o u t h exudate Fat body M a l p h i g i a n tubule Salivary gland L a b i a l gland Hemolymph

Stage' L5 L3 L5 A A A

—

L5 L5 L5 L5 L5 L5 A L - a s c o r b a t e deficient d i e t L5 L3" L5 e

i,-Ascorbate Content" 86 ± 84 69 ± 10 48 ± 40 41 ± 30 63 ± 8 60 ± 14 43 ± 3 39 ± 8 27 ± 11 22 ± 15 15 ± 4 < 1 (4) < 1 (3) < 1 (2)

Ascorbic Acid - American Chemical Society

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