Heterochrony in cotton bollworm, Earias vitella (Fb) following ethoxyprecocene administration

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1 ndian Journal of Experimental Biology Vol. 41, April 2003, pp Heterochrony in cotton bollworm, Earias vitella (Fb) following ethoxyprecocene administration rfan Ahmad Khan & Krishna Kumar* Department of Zoology, University of Allahabad, Allahabad , ndia Received 11 March 2002; revised 5 February 2003 Administration of a synthetic precocene analogue, 7-ethoxy-6-methoxy-2,2-dimethyl chromene to fourth and last instar larvae of E. vitella results into heterochrony, viz. prothetely and metathetely. These disturbances are due to interference with the endocrine system and application of juvenile hormone to treated larvae abolishes the effect of ethoxyprecocene. Novak has described different types of heterochrony in insects, viz. prothetely, metathetely and hysterothetely. These abnormalities arise due to disturbance in the normal ratio between the extent of development of larval and imaginal body parts during metamorphosis and are obviously caused due to interference with the endocrine system. Naturally occurring precocenes and isolated from the bedding plant, Ageratum houstonianum, selectively destroy the corpora allata and reduce juvenile hormone titre Bowers 4 reported several synthetic analogues of natural precocenes and the most active of these compounds was 7-ethoxy-6- methoxy-2,2-dimethyl chromene. This synthetic analogue of precocene was named precocene or ethoxyprecocene and it was found more active than natural precocenes in Locusta migratoria 5 Feyereisen et al. 6 reported that in viviparous cockroach, Diploptera punctata, precocene was more effective than precocene in inhibiting juvenile hormone synth~sis by the corpora allata. n many hemimetabolous insects, precocene causes precocious metamorphosis and degeneration of corpus allatum 7. 1i but this compound has not been found active in holometabolous insects, for instance, in lepidopteran Manduca sexta l2 n the present communication, different types of heterochrony produced as a result of administration of precocene to the cotton bollworm Earias vitella, which is a pest of cotton and lady's finger or okra (AbeLmoschus esculentus), are described. Materials and Methods Earias vitella (Fb) (Lepidoptera: Noctuidae) was reared in the laboratory on fresh seeds of okra *Correspondent author Phone : (R) kkumaruniv@rediffmail.com (Abelmoschus esculentus) at 27 ±) 0c. The first and second larval instars have a life span of about 3 days each. The third instar lasts for a little more than 2 days, viz. about 60 hr whereas fourth and fifth larval instars have a life span of 2 days each. After living for 2 days, fifth instar larva stops feeding and passes through prepupal stage lasting for 2 days and spins a cocoon. After 24 hr of cocoon formation, larval-pupal ecdysis takes place. The duration of pupal instar is about 8-9 days, at the end of which moths emerge out. The synthetic precocence analogue, 7 -ethoxy-6- methoxy-2,2-dimethyl chromene (ethoxyprecocene or precocene ; Sigma Chemical Co., U.S.A) was dissolved and diluted in acetone to obtain different concentrations of the compound in 1 acetone. Treatment of larvae Topical treatment - Fourth instar and fifth instar larvae ( and 2 day old) were treated with 50, 100, 150 and 200 lg precocene in 1 acetone applied on the dorsal surface of posterior abdomen with the help of a Hamilton syringe. Control larvae were treated with 1 pure acetone only. Daily treatment-acetone (1111) containing 50 lg precocene was topically applied on the posterior abdomen of 1 day old fourth instar larvae and this dose was administered daily to larvae throughout fourth instar and fifth instar till the end of prepupal stage. Similarly 1 day old fifth instar larvae were treated with 50 lg compound and this dose was administered daily to larvae till the prepupal stage. Control larvae were similarly treated with 1 pure acetone only. Oral administration - The fourth and fifth instar larvae ( and 2 day old) were starved for a few hours.

2 KHAN & KUMAR: HETEROCHRONY N EAR/AS FOLLOWNG ETHOXYPRECOCENE ADMNSTRATON 329 n separate set of experiments, 1 J.! acetone containing 100 and 200 J.!g precocene was injected into fresh okra seeds and these were fed to starved larvae. When the larvae had completely consumed the treated seeds, they were also fed with fresh untreated seeds. Control larvae were fed with pure acetone treated seeds. JH reversal experiment-juvenile hormone (cis-1o,11-epox y-3, 7, -trimethy trans,trans-2,6-dodecadienoic acid methyl ester; Sigma Chemical Co., U.S.A.) was dissolved and diluted in acetone to obtain 10 and 40 J.!g in 1 J.! acetone. n separate set of experiments, 1 day old fifth instar larvae were topically treated with precocene (100 J.!g) + JH (10 J.!g) and precocene (l00 J.!g) + JH (40 J.!g). Results Topical, daily and oral administration of precocene to fourth and fifth instar larvae of E. vitella resulted into heterochrony, viz. prothetely and metathetely (Tables 1-4). Prothetely- Prothetely consisted of precocious pupae and adultoids. Precocious pupae - Precocious pupae were grouped into different grades according to the extent of development of larval/pupal characters.. Larva transformed into pupa without secreting cocoon or with less secreted imperfect cocoon (Figs 1 and 2); cephalic swelling and larval exuviae clearly visible.. Pupa consisting of highly condensed and compact abdomen; bifurcated proboscis; rudimentary unpigmented eye balls and cylindrical wings (Fig. 3). These pupae were either without cocoons or with imperfect less secreted white cocoons.. Pupa larval in appearance with larval head and extremely reduced thoracic legs (Fig. 4). V. Pupa exhibiting larva like body but having pupal pigmentation; anterior membranous part with light brown pigmentation and rest of the body chitinized with dark brown pigmentation; larval thoracic and abdominal legs present (Fig. 5). Table - Topical application of ethoxyprecocene to fourth instar larvae of E. vitella Age of larvae Dose No. of animals Mortality Prothetely Metathetely (days) (~g) treated (0/0 ) (0/0 ) ( 0/0 ) Coefficient of correlation (r) : , between dose and prothetely; , between dose and metathetely in case of day old larvae; +, between dose and prothetely; -1, between dose and metathetely in case of 2 day old larvae Table 2 - Topical application of ethoxyprecocene to fifth instar larvae of E. vitella Age of larvae Dose No. of animals Mortality Prothetely Metathetely (days) (~g) treated (0/0) ( 0/0 ) (0/0 ) SO Coefficient of correlation (r) : , between dose and prothetely; , between dose and metathetely in case of day old larvae; , between dose and prothetely; +0.95, between dose and metathetely in case of 2 day old larvae

3 330 NDAN J EXP BOL, APRL 2003 V. Miniature pupae were produced when fourth instar larvae were daily and orally treated with precocene. These larvae, instead of being transformed into normal fifth instar larvae, resulted into extremely contracted miniature larva like pupae (Figs 6-8; cf normal fourth instar larva, Fig. 9). Adultoids-Adultoids were classified into different grades depending upon the extent of differentiation of imaginal characters. 1. Adultoid with coiled proboscis; pigmented eye balls; reduced segmented antenna developed only on one side; legs in the form of protuberances; wings absent; abdomen larval in appearance (Fig. 10).. Adultoid with expanded and deformed wings; all the three pairs of legs (prothoracic, mesothoracic and metathoracic) in the form of protuberances; eye balls reduced, the left eye ball considerably smaller than the right one; antennae and labial palpi not developed; proboscis spirally coiled; abdomen unsegmented and without scales (Fig. 11).. Adultoid with wings and other body parts highly deformed; uncoiled proboscis and only two pairs of legs developed (Fig. 12). V. Adultoid with bifurcated proboscis; deformed legs and crumpled wings: fore wings without characteristic green bands (Fig. 13). Metathetely-Metathetely consisted of supernumerary larvae, ecdysial stasis and larval-pupal intermediates. Supernumerary larvae having loose cuticle all over the body and without characteristic larval pigmentation, swallowing of air associated with fledging (ecdysis) and rupturing of abdomen at the posterior end clearly visible (Fig. 14).. Ecdysial stasis - Larva unable to moult into next instar and thus exhibiting total ecdysial stasis; loose cuticle all over the body surface and characteristic white streaks and black pigmentation missing (Fig. 15) or larva with exuviae attached to thoracic legs and posterior extremity of the body showing partial ecdysial stasis (Fig. 16).. Larval-pupal intermediate consisting of pupal body with membranous wing developed only on one side, other pupal characters absent; larval mandibles and pro legs visible; larval exuviae attached to posterior extremity of the body (Fig. 17). Topical, oral and daily administration of precocene to fourth instar larvae resulted into all grades of Table 3 - Oral treatment of fourth and fifth in star larvae of E. vitella with ethoxyprecocene Age of larvae Dose No. of animals Mortality Prothetely Metathetely (days) (J.,lg) treated (%) (%) (%) Fourth instar Fifth instar Coefficient of correlation (r) : + 1, between dose and prothetely; -, between dose and metathetely Table 4 - Fourth ins tar Daily treatment of fourth and fifth instar larvae of E. vitella with 50 J.,lg of ethoxyprecocene No. of animals Mortality treated (%) Prothetely (%) 70 Metathetely (%) 10 Fi fth ins tar

4 KHAN & KUMAR: HETEROCHRONY N EARlAS FOLLOWNG ETHOXYPRECOCENE ADMNSTRATON ~ D. ~_ Figs (,2) - Precocious pupae of grade ; (3)-Precocious pupa of grade ; (4) - Precocious pupa of grade ; (5) - Precocious pupa of grade V; and (6,7) - Precocious pupae of grade V_Bar represents 1 mm.

5 332 NDAN J EXP BOL, APRL r i! m- ~.... _ ml ---= ~~. Figs (8) - Precocious pupae of g rade V; (9) - Normal fourth in sta r la rva; (10) - Adultoid o f g rade ; ( ) - Adultoid of g rade ; (12) - Adultoid of grade and (13) - Adultoid of g rade V. Bar re presents mm.

6 KHAN & KUMAR : HETEROCHRONY N EAR/AS FOLLOWNG ETHOXYPRECOCENE ADMNSTRATON 333 Figs (14) - Supernumerary larva; ( 15 ) - Larva s howing complete ecdysial stasis; ( 16) - Larva showing partial ecdy sia l stasis and (17) - Larval-pupal intermediate. Bar represents mm.

7 334 NDAN J EXP BOL, APRL 2003 prothetely and metathetely, as described earlier, but adultoid of grade and precocious pupae of grade V were not produced with topical appiication. Topical, daily and oral treatment of fifth instar larvae with precocene did not produce adultoids of grade and and precocious pupae of grade V and V whereas all other grades of prothetely and metathetely were formed. Control or acetone treated larvae did not show any developmental abnormalities in any of the treatment. When fifth instar larvae were simultaneously treated with 100!lg precocene and 10!lg JH, it resulted into normal development except that the treated larvae formed white less secreted imperfect cocoons. However, when larvae were administered 40!lg JH alongwith 100!lg precocene, no developmental abnormalities were observed. Discussion Hardie lo reported that ethoxyprecocene was more potent than precocene and in three species of aphids viz. Acyrthosiphon pisum, Aphis fabae and Megoura viciae. Prenatally applied precocene had no effect on the development, precocene induced only supernumerary moult (metathetely) in M. viciae whereas ethoxyprecocene induced both prothetely (precocious development) and metathetely in all the three species of aphids. As compared to the results of ethoxyprecocene in hemimetabolous insects, this synthetic precocene analogue was unable to affect the development of lepidopteran, Manduca sexta l2 The spotted boll worm, Earias vitella is found to be extremely susceptible to ethoxyprecocene as a spectrum of abnormalities (different grades of prothetely and metathetely) are manifested after administration of the compound to penultimate and last larval instars. The fourth or penultimate instar larvae of E. vitella are more susceptible to ethoxyprecocene as compared to last instar larvae as the treatment of former produces higher toxicity and all types of prothetely and metathetely. The greater sensitivity of fourth instar larvae to ethoxyprecocene is possibly due to higher JH titre because in a number of lepidopteran insects, the JH titre is reported to be high during fourth stadium and during apolysis of the last stadia and then it declines before cocoon spinning and again a JH peak. b d d S 0 serve unng prepupa stage.. Administration of ethoxyprecocene to E. vitella larvae results into inhibition of the secretory activity of the labial glands as many precocene treated larvae were unable to secrete cocoons and pupated directly without cocoons or with less secreted imperfect cocoons. n many lepidopteran insects, the labial glands remain inactive during the first to penultimate larval instars but in the last larval instar these become active producing a large quantity of silky material for spinning the cocoons. Since the secretory activity of the labial glands occurs in the presence of JH, its suppression in precocene treated larvae occurs due to JH deficiency. When 10!lg JH is administered to precocene treated larvae, it partly compensates for JH deficiency as the larvae are still unable to fully secrete the cocoons. However, when JH is supplied in appropriate quantity (40!lg), the precocene treated larvae spin the normal cocoons. The prothetely or precocious development is considered to be a true anti JH effect of ethoxyprecocene and has been reported in many hemimetabolous insects viz. aphids where it is due to JH deficiency, as found in E. vitella, and occurs due to destruction of corpora allata l 1.l 5. n such cases there is an acceleration of metamorphosis and the treated larvae of E. vitella tend to acquire pupal/imaginal characters well in advance. For example, treated fourth instar larvae skip the normal development passing through the last stadium and transform into extremely contracted larva like miniature pupae. n E. vitella, prothetely comprised precocious pupae and adultoids of various grades. On one hand, only the secretory activity of the labial glands of treated larvae is either inhibited or suppressed resulting into pupae without or with imperfect cocoons and on the other hand, whole larval body responds and is transformed into miniature pupae due to JH.deficiency as a result of precocene administration. Similarly adultoids of different grades are formed depending upon the degree of differentiation of imaginal characters. This is due to the difference in the intrinsic level of JH in treated insects; lower is the JH titre, higher is the differentiation of pupal/imaginal characters. n E. vitella, the metathetely comprised supernumerary larvae, ecdysial stasis and larval-pupal intermediates. The various metathetelic effects produced are also due to difference in intrinsic JH level in treated insects as a consequence of precocene administration. Ecdysial stasis occurs due to disruption of moulting process and larvae exhibiting total ecdysial stasis naturally has higher intrinsic JH titre as compared to those suffering from partial ecdysial stasis. Similarly in supernumerary moults, whole organism suffers from total juvenilizing effect whereas in larval-pupal intermediates, only a part of the body

8 KHAN & KUMAR: HETEROCHRONY N EARAS FOLLOWNG ETHOXYPRECOCENE ADMNSTRATON 335 suffers from such an effect. Therefore, intrinsic JH level in insect's body determines whether it would form supernumerary larval instar or larval-pupal intermediate or undergo normal pupation. n other insects such as Rhodnius prolixus 8 and Locusta migratoria 16, precocene administration results into metathetely such as supernumerary larvae and ecdysial stasis. These effects are due to deficiency of juvknile hormone because in Rhodnius prolixus, treatment of allatectomized larvae with juvenile hormone reverses the ecdysial stasis 17 n most lepidopteran insects, JH titre is high during the early part of the last stadium and then it declines sharply and again a high JH titre is observed during prepupal stage 18 n Spodoptera littoralis 19 and Mamestra brassicae 20, application of juvenile hormone to last instar larvae shortly before pupation stimulates the prothoracic glands to produce ecdysone. Therefore, a high prepupal JH titre is necessary for successful pupation in lepidopteran insects and its suppression either by allatectoml 1 or by application of antijuvenile hormone compounds like fluoromevalonolactone 22 disrupts or delays metamorphosis. n E. vitella, the metathetely may be due to JH deficiency or suppression of prepupal JH titre as a result of precocene administration, as happens with the application of other anti JH compounds like FMev, and exogenous application of JH to precocene treated larvae abolishes the effect of precocene. Acknowledgement The authors are grateful to Prof. U.S. Srivastava for valuable suggestions and helpful criticisms. Financial assistance from the Department of Science and Technology, New Delhi, is gratefully acknowledged. 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