Indian Journal of Experimental Biology Vol. 37, April 1999, pp. 379-383

Juvenilizing effects of ethoxyprecocene in a lepidopteran insect

Sarika Srivastava & Krishna Kumar*

Department of Zoology, University of Allahabad, Allahabad 211002, India

Received 31 July 1998; revised 24 December 1998

Administration of a synthetic precocene analogue, 7-ethoxy-6-methoxy-2, 2-dimethyl chromene (ethoxyprecocene) to fifth and last instar larvae of Spodoptera litura causes juvenilizing effects such as prolongation of life of penultimate and last larval instar and subsequent postponement of pupation, ecdysial failure, formation of abnormal pupae and larval-pupal intermediates. These effects can be reversed by juvenile hormone application .

Since the discovery of two naturally occurring preco­cenes (I and II) by Bowers et al. I, attempts have been made to produce their synthetic analogues. One such compound, 7 -ethoxy-6-methoxy-2, 2-dimethyl chromene or ethoxyprecocene, synthesized by Bow­ers2, has been found more active than natural preco­cenes in Locusta migratoria3

• The effect of ethoxy­precocene has been widely reported in hemimetabo­lous insects where this compound not only produces juvenile hormone antagonistic effects such as preco­cious metamorphosis but also juvenile hormone ago­nist effects like supernumerary moults and ecdysial stasis3

-8

. Although ethoxyprecocene is not very active in holometabolous insects particularly Lepidoptera9

,

it was found that in Sarcophaga ruficornis (cyc1or­rhaphous Diptera), administration of ethoxyprecocene to last instar larvae caused significant reduction in larval life, precocious pupariation and shedding of larval cutic1e 1o

. In the present communication, juve­nilizing effects of ethoxyprecocene in a lepidopteran, Spodoptera litura . are described.

Materials and Methods Treatment of larvae--Larvae of S. litura were

reared in the laboratory at 27°± 1 °C on castor leaves . Ethoxyprecocene (7 -ethoxy-6-methoxy-2, 2-dimethyl chromene; Sigma Chemical Co. , USA), was dissolved and diluted in acetone to obtain 50, 100, 150 and 200l-1g of the compound in 11-11 acetone. Fifth instar (1 and 2 day old) and sixth instar (1 , 2 and 3 day old) larvae were topically treated with different concen­trations on the dorsal surface of posterior abdomen

*Correspondent auth or

with the help of a Hamilton syringe. Control larvae of similar age were treated with 11-11 of pure acetone only.

For daily treatment, 11-11 acetone containing 50l-1g of ethoxyprecocene was topically applied to I day old fifth instar larvae and this dose was administered daily to larvae throughout fIfth and sixth instar till pupation. Application of ethoxyprecocene to sixth instar larvae followed the same protocol. I day old sixth instar larvae were treated with 50l-1g compound and this treatment was continued daily throughout the last larval instar till pupation . Controls were similarly treated with pure acetone only.

For oral administration, experimental fifth instar (I and 2 day old) and sixth instar larvae (I, 2 and 3 day old) were starved for a few hours. Acetone (11-11) containing 200l-1g ethoxyprecocene was topically ap­plied on a small piece of castor leaf and acetone was allowed to evaporate. It was then given to a starved larva for feeding. When the larva had completely consumed the treated piece of leaf, it was also given fresh untreated castor leaves for feeding. Control lar­vae were similarly fed with small pieces of castor leaves treated with pure acetone only.

Ju venile hormone (JH) reversal experiment - Ju­venile hormone (cis-IO, II-epoxy-3,7, ll-trimethyl trans, trans-2, 6-dodecadienoic acid methyl ester; Sigma Chemical Co. , USA), was dissolved and di­luted in acetone to obtain 10 and 40l-1g of hormone in 1 1-11 of solvent. In separate set of experiments, 3 day old sixth instar larvae were topically treated with eth­oxyprecocene (200l-1g) + JH (1 0l-1g) and ethoxypreco­cene (200l-1g) + JH (40l-1g) .

380 INDIAN J. EXP BIOL., APRIL 1999

Results Administration of 50 and IOO)..lg of ethoxypreco­

cene did not produce any effect whereas highe:r doses

of ISO and 200)..lg (topical application), daily treat­

ment and oral administration of the compound to fifth

and sixth instar larvae produced toxicity and a num­

ber ofjuvenilizing effects which were as follows :

I) Larval-larval ecdysis-When fifth instar lar-

vae were trea ted with ethoxyprecocene (topica l, daily and oral admini stration), it resulted into prolongation

of larval life and consequent delay in lan;al-Iarval

ecdys is except in case of oral treatment of I day old larvae (Tables \ , 3 and 5).

2) Larva/-pupal ecdysis-Topical , dai ly and oral admini stration of ethoxyprecocene to fifth and

six th in star larvae resulted into significant postpone­

ment o f pupati on or prol ongati on o f larval life of last in star larvae except in case o f ora l admini strati on of

the compound to fi ft h in star la rvae (Tables 1-3 and 5).

Application of 200 ~lg o f ethoxyprecocene along­

wi th \ O)..lg JI! to 3 day o ld last instar la rvae resulted into nomla l deve lopment except larva l-pupal ecdys is

was s ignificantly delayed (P < 0 .00 I , I test). How­ever, whcn the J H dose was increased to 40 )..lg,

trea ted larvae pupated alongwith the controls and de­

veloped normally Crable 4).

3) £cd)'sill / /ai /lire-At a higher dose of 200)..lg (toplcdl applIcatIOn ), dall y and oral admin istrat ion of

ethoxyprecocene . trea ted in sects were unable to shed o il fragment s of larval ex uvi ae which were retained

in dIfferent parts of the body in varying degrees. In

some cases, body was ex tremel y con tTac ted acq uiring brown pupal pIg menta ti on but unabl e to cast o fT lar­va l integument (figs I and 2; Tables \- 3 and 5).

4) A hnorlll ([/ jJ lIpae- A bnormal pupae prod uced as a conseq uence of e thoxyprecocene admin istration , consi sted of bifurca ted probosci s; wi ngs hi ghl y re­duced and swo ll en at the tips; antennae showin g sign of segmentation: body having usua l brown pigmenta­ti on but the re was a white membranous patch o f in­tegumen t devoid of tannlllg on the ventra l side o f an­te ri or abdo men: pupa l cremaster reduced (Fi g . 3; Ta­bles 1-3 and 5)

5) Lu /' \'(// -pll/)(I / interlll ed ia tes- Topical applica-tIOn of cthox yprecocenc to 2 day o ld fifth and s ixth instar larvae (Tabks I and 2) and ora l admini stration o f the co mpou nd to 2 day o ld fi fth and 3 day old sixth instar larvae (Tab k 5) resulted into form ation o f lar-

val-pupal intermediates consisting of anterior part of

the body larval in appearance whereas posterior half was transformed into pupa (Fig. 4) .

Discussion Administration of ethoxyprecocene to fifth and last

instar larvae severely disrupts moulting and meta-

i

j

rJ

Fig. I- Larva wi th exuv iae from posterior hal f of th e body not shed (ecdys ial fa ilure) Fi g. 2-Larva wi th ex tremely contrac ted body acqu iring brown pupal pig mentation hut un ab lc to ccd ysc. Fi g. 3- Ab normal pupa. Fi g. 4----Larva l-pupa l intcrmcdiatc .

Age of larvae (days)

Control

Control 2

Control

Control

ISO

200

ISO

200

SRI V AST A V A & KUMAR: JUVENILIZING EFFEcrs OF ETHOXYPRECOCENE

No. of animals treated

20

10 20

10 20

10 20

10

Table I-Topical application of ethoxyprecocene to fifth instar larvae of S. litura

% mortality

SO

30

30

40

Days required for larval-larval ecdysis

(Mean± SD)

3.8±O.4 (P<O.OO I, I test)

3tO 4tO

3tO 4.8 to.4

(P<O.OO I , I test) 3tO 4tO

3tO

Days required for larval-pupal ecdysis

(Mean ± SD)

7.6±O.48 (P<O.O I , I test)

6.4 to.48 7.5 t 0.5

(P<O.OO I , I test) 6tO

7.57 to.49 (P<O.OO I, I test)

6tO 6.5 to.S

(P <0.02, I test) 5.6 to.48

Normal pupae %

50

100 20

100 50

100 40

100

Abnormal pupae %

10

10

Ecdysial failure %

50

10

• A t SO and 100~g, no effect was observed.

Age of larvae (days)

Control

Control 2

Control

Control 3

Control

Control

Dose· (Ilg)

150

200

150

200

150

200

Table 2-Topical application of ethoxyprecocene to sixth instar larvae of S. litura

No. of animals treated

20

10 20

10 20

10 20

10 20

10 20

10

% mortality

40

20

40

10 40

40

10 40

Days required for lar­val-pupal ecdysis

(Mean± SO)

5.66 ± 0.47 (P<O.OI , t test)

4.8 ± 0.4 5.5 ± 0.5

(P<0.02, t test) 4.8 ± 0.4

6.33 ± 0.46 (P <0.00 I , t test)

5±0 6.33 ± 0.46

(P <0.00 I, t test) 4.8 ± 0.4-

6.16 ± 0.37 (P <0.00 I, t test)

5±0 6.S ± 0.5

(P < 0.001 t test) 5±0

Normal pupae %

60

100 50

100 100

90 40

100 100

90 20

100

Abnormal pupae %

30

10

Ecdysial failure

%

10

30

• At 50 and 1001lg, no effect was observed.

Table 3--Daily treatment of fifth and sixth instar larvae of S. litura with 50llg of ethoxyprecocene

No. of % Days required for Days required for Normal Abnormal animals mortality larval-larval ecdysis larval-pupal ecdysMi pupae % pupae % treated (Mean ± SO) (Mean ± SO)

Fifth instar 30 30 4±0 7.6 ± 0.48 IV 20 (P <0.001, t test)

Control 10 3±0 6±0 100 Sixth instar 30 40 5.33 ± 0.46 30 10

(P <0.05, t test) Control 10 4.6 ± 0.48 100

381

Larval-pupal intermediate

%

10

Larval-pupal intermediate

%

10

Ecdysial failure

%

40

20

382 TNDIAN J. EXP BIOL., APRIL 1999

morphosis in S. litura. When applied topically, lower doses of 50 and 100Jlg of the compound do not pro­duce any effect whereas various abnonnalities were observed at higher doses of 150 and 200J.1g. Oral ad­ministration of the compound is less effective in de­laying larval-pupal ecdysis as compared to topical and daily treatment but causes severe disruption of metamorphosis .

Administration of ethoxyprecocene to larvae of S. filum leads to JH agonist effects such as prolongation of life of fifth and last instar larvae or postponement of pupation , ecdysial failure, inhibition of tanning and formation of abnonnal pupae and larval-pupal inter­mediates which are also produced by the administra­tion of juvenoids ". This is in contrast to the effects of ethoxyprecocene in another holometabolous insect, Sarcophaga ruficornis (cyclorrhaphous Diptera) where it produces JH antagonistic effects. like signifi­cant reduction in life. of last instar larvae, precocious

pupariation and ecdysis of larval cuticle (destined to fonn puparium) and these effects can be reversed by JH application 10. Edwards et al.9 have found that in lepidopteran, Manduca sexta, antijuvenile honnone compounds such as FMev (tetrahydro-4-fluoro­methyl-4-hydroxy-2H-pyran-2-one) and ETB (ethyl-4-[2-(tert-butylcarbonyloxy) butoxy]-benzoate) not only produce symptoms of JH deficiency but also significantly reduce titre of endogenous juvenil.e honnone whereas application of ethoxyprecocene does not produce any effect. They have offered no explanation why this synthetic precocene analogue is not active in this insect species. Similarly in Tricho­plusia ni (Lepidoptera), anti-JH compounds like FMev and DPH (3, 3-dichloro-2-propenyl hexanoate) disrupt metamorphosis causing delay in tanning and formation of abnormal pupae and larval-pupal inter­mediates resulting from reduced JH levelsl2. Various JH agonist effects of ethoxyprecocene in S. fitum, are

Table 4--Juvenile honnone(JH) reversal experiment

Age of larvae

(days)

3 (S ixth in sta r)

Control

Con tro l

Dose

Et hoxyprccocene

(200pg)+ JH ( I Opg)

Ethox yprecocene

(200pg)+ JH( 40pg)

No . of animals % treated mortality

20 10

10 20 10

10

* No abnormal pupae, ecdysia l fa ilure or larval-pupa l intennediates were observed .

Days required for larval - Nonnal pupae·

pupal ecdysi s % (Mean ± SD)

7.25 ± 0.43 90 (P <O.OO I, I test)

S ±O 100 5±0 90

5±0 100

Table 5--Dral treatment of fifth and six th instar larvae of S. filUm with 200 Ilg of ethoxyprecocene

Age of No . of (Yo nlor- % larvae fail ed Days required for Days required for Normal Abnormal Ecdysial Larval-larvae animal s 13lity to undergo larval-larval larval:pupal pupae % pupae % failure % pupal (days) treated larval-larval ecdysis ecdysis intermedi-

ecdys is (Mean± SO) (Mean ± SO) ate % Fifth ins ta r I 20 30 10 3. 11 ±0.31 7.16± 0.89 20 20 20

(P >0. 10, ( test) (P >0. I 0, ( test) Contro l 10 3 ±0 66 ± 048 100 2 20 40 4.8 ± 04 7.16± 0.89 10 30 20

(P <0.001 , (test) (P >0.1 0, ( test) Control 10 3±0 7.6 ± 048 100 Sixth in star

20 30 7. 14±O.63 60 10 (P<O.OO I, ( test)

Contro l 10 54 ± 048 100 2 20 50 6.6 ± 048 10 40

(P <0.0 I , ( test) Contro l 10 5.4 ± 048 100 3 20 50 6.8 ± 0.74 10 20 20

(P <005. ( test) Control 10 6± 0 100

SRIVASTAVA & KUMAR: JUVENILIZING EFFECTS OF ETHOXYPRECOCENE 383

similar to those produced by application of FMev, ETB or DPH in other lepidopteran insects. These ef­fects are certainly due to juvenile hormone deficiency because application of JH to last instar larvae treated with ethoxyprecocene results into normal metamor­phosis in a dose dependent manner. It has been re­ported that this synthetic precocene analogue causes degeneration of corpora aHata and inhibition of juve­nile hormone secretion in adult cockroach, Dip/op­tera punctata l.1 and in the embryos of Locusta migra­toria I4

•15

.

In most lepidoptera, JH titre is high early in the last stadium and then it continues to be low except for a brief peak of JH in prepupa 16. IS. In Spodoptera litto­ralis , application of. JH to last instar larvae shortly before pupation stimulates prothoracic glands to pro­duce ecdysone resulting into acceleration of meta­morphosisl 9

. Therefore, it is likely that prepupal JH peak in ethoxyprecocene treated larvae of Spodoptera litura is either delayed or suppressed and the de­creased JH level may not be sufficient .for activation of prothoracic glands and thereby leading to effects similar to those produced by exogenous administra­tion of juvenoids. In Rhodnius prolixus also, delay in moulting after precocene treatment can be reversed by JH applications.

Acknowledgement The authors are grateful to Prof U.S. Srivastava,

Former Professor and Head of the Zoology Depart­ment, University of Allahabad, for encouragement and helpful suggestions. Financial assistance from

Department of Science and Technology, New Delhi , is gratefully acknowledged .

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