Indi an Jou rnal of Experimental Biology Vol. 38, February 2000, pp. 137- 144

RH-5992-An ecdysone agonist on model system of the silkworm Bombyx mori

V S Kumar1, M Santhi2 & M Kri shnan 1

1Departmcnt of Biotechnology. Bharathidasan University, Tiruchirappalli 620 024, lndi:1 2Departmcnl of Biochemistry, Seethalakshmi Ramaswamy college, Tiruchirappalli 620 002. lndi:1

Received 4 Jun e !998; revised 6 September 1999

RH-5992 is a novel synthetic non-steroidal ecdysteroid agonist with a high selecti vi ty towards Lcpido pt er:1n species . The effect of ecdysone agonist RH-5992 on larval peri od, larval weight , si lk gland weight and hacmolymph protein profil e were examined in the model organi sm, the larvae of silkworm, Bombyx mori . The LD 511 values were found to he I 6.21 and 12.01].lgliarva for 72 and 96 hr respectively. In the present study, three subl ethal conccmralions of l/5 'h. IIIO'h and l/20'h of LD111 at 72 hr were selected and applied on the mid-dorsa l line of the silkworm B. mori. The maximum monal it y of ]5'/r wa' observed in the group which received the highest (3.2 ].lgliarva) concentration of RH-5992. The mortality rate was found to be dose dependent as well as time dependent. Interest ing results were observed in haemolymph prolilc of the Rl-1 -5'.!92 treated larvae as staining intensity of 30kDa protein decreased sign ilicantly whereas the effect was not marked 011 olhcr 111:1-jor proteins like storage proteins and vitellogenin polypeptides. From the results, it is confirmed th at RH-5'.!'.!2 causes changes in larval characters and protein prolile of silkworm B.11wri ll is proposed th at RH-5992 may cause negati ve ellcct speci11call y on reproductive characters like development of ovary and egg production due to decrease in JOkD:1 protein .

The indi scriminate use of pesticides have caused ir­reparable damage to the ecosystem in many places of the world. The wide applicability of pesticides threat­ens the survival of non-targeted beneficial organisms. During the past few decades organic biocides have been found to be increasingly important in controlling pests in and around mulberry fi e lds. By feeding the silkworm with such contaminated mulberry leaves, various effects such as premature moulting, deterio­rated cocoon quality , reduced cocoon yield and eventual death often occur 1 therefore, this situation augments the study of the effects of pesticides in the model organism with spec ial refe rence to silkworm

growth and physiology of the silk production . The non-steroidal ecdysteroid agonist , RH-5992,

has been shown to act similar to ecdyste roids in initi­ating precocious moulting in LepidopteransD . RH -5992 mimics the moulting hormone 20-hydroxy­ecdysone and compete e ffec tive ly for binding to ec­dysteroid receptors and is 10 times more active than the natural hormone and appears to pe rsist in the epi­dermis much longer than 20-hydroxyecdysone4

.

Because of the ir physiolog ica l significance, the ecdysteroid-endocrine system, offers a complete panel of potential targets for pes t management strate­gies which also have the additional advantage of be­ing unlike ly to affect vertebrates. RH-5992, a closely

related compound, has undergone considerabl e fi e ld eva luation and has proven to be a ve ry effic ien t con­trol of Lepidopte rans in orchard s, vineyards, vegeta­ble fi e lds and forestss

The effect of such analogues on in sec ts is vari ed .

Winget a/.,6 were able to it;duce pre mature initiati on of moulting at all larval stages in tobacco horn worm, Manduca sexta with RH-5 99f. . They ha ve po inted out that the effects can be characterized hy pathophys i­o logical sy mptoms of hype recdysonism. It has been shown to cause neurotox ic side effects7

Here we extend the study on the e ffect of ecdysone agonist RH-5992 on the ph ys io logy and biochemistry of the economically impo rtant , non-targe t Lepidop­teran , silkworm, B()/nbyx mori.

Materials and Methods Experimental animals- Silkworm larvae, H. 111o rt

(cross breed : L. a multivoltine , X NB4 D ~ a hi vo ltine) were reared on mulberry leaves at 2:i ° C 6) ± Y !r relative humidity and a photoperi od o r 12: 12 hr li ght and darkness. Newly ecdysed 51

h inst~u· larv~ t e were used for the present s tudy.

In vivo assay for the effects of RH-5992 Application rd' Rf-/-59{}2-From the stock so luti on

of RH-5992 ( I mg/2ml of acetone), various concen-

138 INDIAN J EXP BIOL, FEBRUARY 2000

!rations were prepared and applied topica lly along the mid-dorsal line of the larvae. Mortality percentage included both dead and affected larvae.

Medial lethal doses were assessed by the Finney 's method of probi t analysis8

. Out of the three groups of larvae, group I were treated with 1/5'11 (3.2 J.lg/larvae), group II with 1/10'" ( 1.6 ).lg/larvae) and group III with

1/20 (0.8 f..Lgllarvae) of the LD50 concentration at 72 hr respectively. Pos iti ve cont ro l group rece ived 10 ).l l of acetone and negat ive control group was maintained without any treatment. Both treated and control larvae were maintained at rearing conditi ons described ea r­lier. The effects were observed dail y after the treat­ment during the fifth instar deve lopment peri od (day2-6).

Haemolymph proteins-Coll ection of haemolymph was done by injuring the abdominal legs of B. mori larvae and haemolymph was bled from the wound directl y into the eppendorf tubes coated wi th thiourea, an anti ox idat ive agent, and centrifuged at a low speed to remove haemocytes. The supernatant was stored in -70 ° C until further analys is .

Electrophoresis of samples-Polyacrylamide gel electrophores is was carri ed out to study the protein profil e of haemolymph during development of both control and treated larvae, in the presence of Sodium Dodecy l Sulphate ( I 0% SDS-PAGE), according to the method of Laemmli 9

. Densities of protein bands were· measured with a microdensit ometer (Hoefer GS 300). The 30kDa proteins were identifi ed by their positions .

Results In vivo effects of RH-5992 on larval dura tion-In

order to study the deve loping larva l characters, sub­lethal concentrat ions were chosen from LD50 va lues whi ch all ows the larvae to su rvive for 6 days. The LD5o va lues determined for 72 and 96 hr were 16.22 and 12.0 I ).lg/l arva respectively. For the present study, 1/5, 1/IO and l/20ofLD,oat72 hrwaschosen.

It was found that the percentage of mortality in­creased in all the treated groups as the time of ex po­sure increased. It was also observed that none of the larvae died in the tes t groups before 48 hr. After 72 hr, the percentage of mortality was 10% & 35 % in the group that received lowest (0. 8 ).lg/ larva) and hi ghest (3.2 ).lg/larva) concentrati on of Rl-1-5 992 respecti vely (Fig. 1). The percentage mortality al so increased with increas ing concentration of RH-5992. Although the larvae of treated groups survived till 6'" day, none of

the larvae li ved to spin the cocoon. Typical phenotyp ic effects induced by Rl-1 -5992

was observed in the prese nt study. The Rl-1-5992 treated larva started showing effects 12 hr after treat­ment when they become quiescent and stopped feed­ing. Within 24 hr of treatment , the head capsule started slipping for the moult. Unlike in normal larvae where head capsul e slippage is foll ovved hy molting, treated larvae remained arresed at thi s ~ t a ge wi thout further development and ecdys is. The treated larvae were simil ar in size and showed le thargic move ment. After 24 hr the colour of the skin chan ged to brown and excreted a semi so lid faeca l matter. The Rl-1 -5992 treated larvae di ed after 6 clays without proceeding to larva l-pupal transformation , unlike the control whi ch completed cocoon spinning in 7 days.

Effects of RH-5992 on lon•ol ,: f/1(1 silkglwul weight- It was observed th at the larva l and si lkgland weight decreased with increas ing concentrati on or RH-5992. Larval weight dec reased dr:11nati ca ll y in the treated larvae from 2'"1 clay of treat men t at hi gh con­centrati ons. Figs 2 and 3 show the (! bse rved l:trva l and silkgland weights of the control an d treated lar­vae at various RH-5992 co ncentrati ons. Ltrval and silkglancl weights showed an increase at low conce n­trati on while they decreased at hi gher co nce ntrati ons of RH-5992. It was found that a negative linear rela­ti onship ex isted between the applied RH-5992 and the silkglancl weight.

Effects of RH-5992 on /wenwl,·nlfJ/1 pmtein ;no­file-To find out the va ri ations in hacrno lymph pro­teins during the larval pe ri od or th e ex periment , anal ys is of the protein profil e was ca rried out dail y during the deve lopment of 5'1r in star larva. In co nt rol

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Fig. !- Larval mortality in control (a: po>i livc co111 rol. h: tll:!'.: ti ve co ntrol) and l :~ rvae treated wi th dirrcrcnt suhklil ;ll conccnlr:t­tion s of Rl-1 -5992 during li fth in slar or si lbvonn . !lolll f,n 111nri.

KUMAR eta/.: RH-5992-AN ECDYSONE AGO 1ST & SILKWOR M 139

larvae, 14 coomassie blue stained bands of molecular weight ranging from 194-14 kDa were resolved by SDS-PAGE ( 10%). The haemol ymph protein profile of the control larvae showed increas ing staining in­tensity of 30 kDa and 80 kDa proteins from day 1-5 of 5'" instar. It was found that the haemolymph pro­tein profile of the RH-5992 treated larvae showed significant decrease in 30 kDa protein during 5'" in­star development. The intensity of 30 kDa protein decreased with increas ing concentration of RH-5992. Quantitative changes in the protein profil e was ob­served in the RH-5992 treated groups by Densi­tometric scanning. The staining intensity of 30 kDa protein in the haemolymph significantl y decreased as

LARVAL WEIGHT(g)

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SUB LETHAL CONCENTRATIONS OF RH-5992

Fi g. 2-Larval weight in control (a: positive control. b: negative control) and larvae treated with di ITerent sublethal concent rati ons of RH -5992 during liftb instar or silkworm, /3ombrx 1/ IO I"i.

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Fig. 3-Sil kgland weigh t in contro l (a: posi live control, b: nega­ti ve con trol) and lnrvae treated with dillercnl sublethal concen tra­ti ons or RH -5992 during lifth in star or si lkworm, /Jombrx 1/IO I"i.

correlated by the percentage dec rease in the scanning area. Maximal percentage decrease was observed at high concentrati on of RH-5992 (Figs 4-8).

Discussion The studies reported here show th at the substituted

dibenzoyl hydrazine, RH-5 992, induces an incom­plete moult in the B. mori larvae. caus ing the imme­diate onset of a moult with the sli ppage or the head capsule within 24 hr and di ed without further deve l­opment and ecdys is. When penultimate in star larvae of Choristoneura .fiuniferana were treated with RH-5992, a new larva l epicuticle was formed but no lar­val late endocuticle was depos ited and the new head capsul e remained thin and untanned 10

. Similar effec ts were seen in the B. mori larvae, treated in thi s stud y, except that the head capsule and mandibl es often showed marginal sc leroti zation.

The morphologica l deformiti es of cuti c le formation are a result of the effects on these compounds on the ex press ion of the genes for cndoc uti cular proteins such as LCP-1 4 and for Dopa DeCarbox ylase (DOC ). the enzyme required for sc leroti za ti on. RH -5992 pre­vented release of ec losion hormone (E H) (A. Retna­karan eta!. , unpubli shed). Inj ec ti ons of ecdysteroids into larvae cause an immediate interrupti on or feed­ing. Gut purge in the last in star larvae or /\!! . . 1exw is preceeded by two small pea ks and associa ted with a di stinct commitment pea k or endogenous ecdyste r­oids11. Terminati on of feed ing is an import ant pre­paratory step of moultin g and it s co ntrol by an in­creased level of ecdysone is probabl y common bu t

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Fig. 4-Percnt age decrease in th e ;1rca or .10 KD;J pn>l cins scan ned after treatmen t wit h dirrcrcnt subletha l conccntr;H iom. or RH-5992 during the lirth insta r dcvclopmc nt;tl pcriud in silk­worm, /Jombyx mori.

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INDIAN J EXP BIOL, FEBRUARY 2000

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Fi g. 5-Densitometri c scanning o r haemolymph po lypeptides or control (a: positi ve co nt ro l, b: negati ve co nt ro l) and I:Jrv;Jc tl·c:ncd with different sublethal concentrati ons or RH- 5992 (c: 1/20 . d: 1/1 0, e: 1/5, of LD 511) on day 2 or firth instar sil kworm. llollll•r.rlllori.

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Fig. 6-Densitometri c scanning o r haemolymph polypeptides or contro l (a: positi ve cont ro l, h: negati ve con trol ) :11H.i la rv:•c trc:ncd wi th different subl ethal concentrations of RH -5992 (c : I /20, d: Ill 0, e: I /5, of LD,0 ) on day :l of firth in star silk worm. l!ondn .1 11 111ri.

KUMAR et a/.: RH -5992-A ECDYSO E AGONIST & SILKWORM 14 1

not genera l. Hi gh concentrati on of 20-hydroxy­ecydysone (HE) have been suggested to cause mor­ta lity due to excess ive haemo lymph ecdys te roid lev­e ls which cannot be metabo li zed or excreted rapidl y (hyper ecdysoni sm) 12

•

The centra l ro le of ecdystero id s in coordinating growth and development during insect postembryonic deve lopment has been well documented 13

. Artific ia l

e levati on of ecdysteroid during earl y stages by treat­ment with 20-hydroxyecdysone, causes CA to e levate JH and an additi ona l larva l-l arva l moulting was in­duced instead of metamorphosis 14

. We suppose that when RH 5992 is applied at hi gh concentrati on, dur­ing earl y last insta r, the CA stimul ating factors (neu­ra l or hormona l) are continua ll y present and effec t direct stimul ati on of CA-ac ti vity. The observed mor­ta lity can be corre lated to thi s hormonal imbalance.

An induction o f s ilk gland degradati on by high 20-

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hydroxyecdysone doses was shown both in sill! and in vitro 1

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. The dec line o f R A and the func ti ona l re­gress ion of silk g lands are primaril y due to the cessa­tion of transc ri ption that is caused e ithe r by lack of nutrients or by high ecdyste ro id titre. T hi s is lin ked to degradati on of cellul ar organe lles o f proteosynthes is that a lso ensures both in the sta rving and pu pa ting

larva 17• On tht other hand , the in vo luti on or s il kg lancl

is a specific response to hi gh ecdys te ro id concentra­ti on acting in the abs(:nce of the JH . Poss ible role of high concentrat ion o f RH-5992 in the dec li ne o f to ta l RNA synthes is and degrad ati o n o f ti ssues have been thought to decrease the la rva l and s il kg land we ights of B. mori.

In the present study majo r haemo lymph prote in s, 30 kDa decreased with inc reas ing co nce ntrat ion o r RH-5992. It has been shown that 30 kDa prote ins a re the other mos t abundant prote ins in the hae mo lymph

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Fig. ?-Densitometric scanning or haemolymph polypeptides or control (a: positi ve contro l, b: negative control ) and l;1 rv;1c trc;ncd \\'ith dirferent sublethal concentratLons or RH-5992 (c: 1/20, d: 1/10, e: 1/5 , or LD511) on day 4 or fifth instar silk worm. Ro111 hr.r nwri.

142 I DIA J EXP BIOL, FEBRUARY 2000

• IS of B. mori at the late larval and early pupal stages . ln the fema le pupae, 30 kDa prote ins are also detect­able in the mature oocytes together with vite llin, a female spec ific yo lk glycolipoprotein 1

'1. Aedes is the

only spec ies known in which ecdysone appeared to " I I . I I " I . ?O ? I ?J I . sttm u ate vtte ogen111 synt 1es ts- ·- ·--_ t ts note wor-

thy that ecdysone stimulated both the fema le spec ifi c and general prote in synthes is in fatbody culture of Bombyx, but egg-spec ific proteins were not synthe­sised. Ecdysone exerts widesp:ead metabolic control of fat body fun cti on, as it is known to stimulate bulk synthes is of RNA and proteins2

.1'24

. The dec rea~e in 30kDa protein , mi ght be related to inhibiti on of pr· -tein synthe~ i s at hi gh concentration of RH-5992 . ll ts shown that there is no maj or changes in other haemolymph proteins in the RH -5992 treated larvae. The present results suggest RH -5992 is spec ifi c to 30kDa protein and further inves ti gat ions arc required to elucidate the effect of RH-5992 on the 30kDa pro­tein gene expression and sy nthesis.

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The substituted dibenzoy l hydraz ine RH-5992 ac ts similarl y to that of ecdysteroid in it s direct act ion on gene express ion earl y in the moulting process and it s suppress ive actions at the end of n~ou lt 4 . The mor­phogeneti c effect is ecclysteroid spec i fi e and ca n be

k 9,, ~ :) 'h evo eel by RH-584 - and also by RJ-I -)l,91 - . at

much lower concentrati ons than 20-hyclroxecdyso nc and RH-5849. The biologica l <h: ttvit y ts RH -5992>>20-hydroxyecdysone>RH-5~49 '7 In additi on to its hormonal effects , RH-5992 1 ~ 1 a y c:1use some neurotox ic side effects7 RH compounds al so inh ibi t I . . I . ?R ?'J B . I . c 11ttn synt 1es ts- ·- . . mort seems to 1c more sensl-

ti ve to the inges ted ecclysteroids than other insect s 12

and thus provides a suit ab le model i'Llr ex pl orin g dil"­ferent effects of ecdysone and 20- hyd roxycccl yso ne. Hence, the use of lepidopteran sriecili c hi oin sec ti cidc RH -5992 main ly near silkworm reari ng ce ntres should be done very cau ti ously. PresL· nt stud y wou ld probably initi ate more understand ing or the-control or sy nthesis of 30 kDa protein spcc il'i c:tl ly hy RH -5991

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K UMAR el a /. : RH -5992-AN ECDYSONE AGONIST & SILKWORM 143

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·I Fig. 9-Densitometri c scann ing of haemolymph polypeptides or contro l (a: pos iti ve control. b: negati ve con trol ) ami L1 r1·:te treated ll'ith different sublethal concentrations of RH -5992 (c : 1/20, d: 1/ 10, e: 1/5, of LD511) on day 6 of fi rt h in star sil kworm. /lnmhr.t 11wri.

to unravel the mysteri es of gene express ion in fat body cell and application of thi s information in de­veloping a strategy for insect control by interfering with the process of molting and metamorphosis. Jn addition to its potential as a powerful , Lepidopteran­specific, pest control agent , its stability relative to that of the ecdysteroids should make it an important tool in the elucidati on of the mode of ecdysteroid ac­tion in the initiation and coordination of the moulting process.

Acknowledgement The authors are grateful to Dr. Tarl ochan S. Dhadi ­

alla, Rohm and Haas Company, U.S .A for the gener­ous gift to technical grade RH-5992. Financial ass is­tance from Internati onal Foundation for Science, Sweden to M.K (B/2520) is acknowledged.

References I K .P Kashi , J Seric Sc i, Japan , 4 1 ( 197 1) 30 I_

2 Retn akaran A, Hi ruma K, Palli S R & Ridd i tord L M , !nsecl Biochem Malec Bio i , ( 1994) 109.

3 Retnakaran A & Oberl ander H, in Colil ro f of ch ilin sylllhesis in insec/s, edited by Muzzarelli R.A.A (European Chitin So­ciety, Lyon) 1993,89.

4 Retnakaran A , Hiru ma K, Pall i S P. Ridd ifmd L M . ln.w·cl

Biochem Mol Bio i , 25 ( 1995 ) I 04.

5 Couilland F, in Rece11 1 adt"lii /U'S i11 in stTI mdncrill <' 1"1' ­

sea rch, edited by D . Muraleedharan and Mari :ttntna Jacob (Associati on for Advancement of Ento tm1lugy. Indi a) I 'J'J5. 14 1.

6 Wing K D, Science, 24 1 ( 19XX) 467.

7 Salgado V L, Peslic Biochem fJhrsinl, .n ( l lJ'J2) I .

8 Finney D J, Probil ana lrlsis (Cambridge Uni vers it y Pre>s. Cambridge) 197 1, 333.

9 Laemmli U K, Na uu·e , 227 ( 1070) (1XO.

I 0 Binnington K & Ratnakaran A . in Epidermis- A hinlogimff_,.

aclive /a rgel f or melllho lic i11 hihi1nrs. ed ited by Hinnington K & Retnakaran A, (Physio logy or Insect Epidenni s. CS IRO. Canberra) , 199 1, 307.

II Wolfgang W J & Ridd iford L M , / Jt-1· !l in!. 11.\ i i 'J X6l .105.

12 Kubol I , Kl ocke J A & A sa no S. J. fl!st•cf fJhrsiol. 29 1 I 'JX3 ) 307 .

13 Smith S L, Regulation of ecdys teroid titre: Synthes i,. in Comprehensive insec/ flhrsiologr . Uiocht'lll isi iT l/1/(1 fJflllr·

macology, ed ited by G A Kerkut and I Gilbert (Oxford . Per-gamon Press) 8, 1985 . 295. ·

14 Gu S H, Chow Y S, .1/nsecl fJhrs io l. 42 ( I 'J%) (J25.

I 5 Chinzei Y , Appl En lom ol Zoo /. I 0 I I 'J75 l 1.-,(J.

16 Kodri k D & Sehnal F. In! ./ Mo rf J/i ol l :.lllhrml. 23 I I 'J -+ l 3 1 I.

17 Blaes N, Couble P& Prudhomme .I C. Ct·ll Ti_,_,·uc· Nt•s. 2 1:1 ( 1980) 3 11 .

I!\ Izumi S & Tommino S, Zoo/ Mog . XS I I 'J7(l) 406.

144 I DIAN J EX P BIOL, FEBRUARY 2000

19 Izumi S, Tojo S & Tomino S , /n sec Biochem , 10 (1980) 429.

20 Hagedorn H H, Fallon A M & Laufer H, Devl Bioi , 32 (1973) 285 .

21 Fallon AM , Hagedorn H.H. Wyatt G R & Lanfer H, J lnsecr Physiol, 20 (1974) 18 15.

22 Hagedorn H, in Comprehet1sive insecr physiologv. biochem­isry and pharmacology, ed it ed by Kcrkut GA& Gilbert Ll (Pergamon Press, New York) 1985 , 205.

23 Price G M, Bioi. Rev, 48 ( 1973) 333.

24 Kress H, Wilhelm Roux's A rcl1s , 182 (1979) 107.

25 Spindler Barth M, Turberg A & Spindler K D. Darch /n .,ccr Hiochem Phvsiol, 16 ( 199 1) I I .

26 Spindler Barth M, Junger E & Spindler K D. Tissue Cl'il. 2-1 ( 192) 9 19.

27 Quack S, Fretz A, Spindler Barth M & Spindler K D. /:·u,. .I Enromol, 92 ( 1995) 34 1.

28 Baumeister R, Ludwig M & Spindler B.trth ivl. Nar urwis.H'/1 - · schajren 79 ( 1992) 185.

29 Spindler Barth M, Kammann V & Sind ler K D. in Chiri11 & Chirosam , edited by Skjak-Brcak G. Arll ho n,on T & Sand­ford P (E lsevier App led Science. London r JlJ))<J . 279 .