Insect Biochem.. Vol. 8, pp. 193 to 195 0020-1700/78/0601-0193502.0010 Pergamon Press L td . 1978. Printed in Great Britain

E C D Y S O N E A N D J U V E N I L E H O R M O N E ACTIVITY IN THE L A R V A E OF THE COCKROACH

P E R I P L A N E T A A M E R I C A N A

ELI SHAAYA Laboratory of Insect Physiology, The Hebrew University, Jerusalem. Israel

(Received 29 June 1977; revised 18 July 1977)

Abstract--Changes in ecdysone and juvenile hormone titre during the 6th larval stage of the cockroach Periplaneta americana were studied. Juvenile hormone is secreted mainly in the second quarter of the instar, while the ecdysone titre mcreases gradually in the third quarter and reaches a maximum shortly before ecdysis. Following this period the titre of ecdysone drops rapidly and it is very low at the time of ecdysis. The increase in ecdysone concentration is correlated to the synthesis of the new cuticle and when the titre of juvenile hormone is at a low level. The maximal concentration of ecdysone and juvenile hormone were similar, about 30-35 ng per gram insect. [n the larval stage, juvenile hormone exerts its effect at the beginning of the instar while ecdysone acts later.

I N T R O D U C T I O N

MOULTING and m e t a m o r p h o s i s are un ique e v e n t s in insec t life. These changes are regula ted by two h o r m o n e s , e c d y s o n e and juven i l e h o r m o n e . The m e c h a n i s m by which these two h o r m o n e s in te rac t in these p roces se s is essent ia l ly u n k n o w n . The t i t re of e c d y s o n e dur ing insec t d e v e l o p m e n t has been s tudied in e n d o p t e r y g a t e o rde r s of D ip t e r ans ~SHAAVA and KARLSON 1965a) and L e p i d o p t e r a (SHAAYA and KARLSON 1965b; BOLLENBACHER et al. , 1975), The pu rpose of th is s tudy is to e x t e n d our k n o w l e d g e to an e n d o p t e r y g o t e insec t b y de t e rmin - ing the e c d y s o n e and juven i l e h o r m o n e levels in a larval s tage. The juveni le h o r m o n e e s t ima t ion was fac i l i ta ted us ing a sens i t ive b ioas say d e v e l o p e d in the c o c k r o a c h (BODENSTEIN and SHAAYA, 1968).

M A T E R I A L S AND M E T H O D S

The insect cultures and all experimental animals were kept in a constant temperature room at 25 °+ - 0.5°C. Cls juvenile hormone (DL-methyl ttt- 10 epoxy-7-ethyl-3,1 l-di- methyl-2.6-tridecadienoate) was used for the study of the effect of juvenile hormone on protocatechuic acid glucoside accumulation in the left colleterial gland (BODENSTEIN and SHAAVA, 1968). The desired hormone concentration was prepared in 2/~1 olive oil. The hormone was injected into the abdomen of a decapitated female. 4-8 hr following the adult ecdysis. The glucoside content of the left colleterial gland was measured 6 days after hormone administration, the mortality rate being about 5%. Details of the structure of the colleterial gland were given earlier by BODENSTEIN and SHAAYA 11968L Protocatechuic acid glucoside con- centration was measured spectrophotometrically accord- ing to the method of WILLIS and BRUNET (1966). The freshly dissected gland was homogenised in 0.4 ml of a saturated aqueous solution of ammonium sulphate to pre- cipitate the proteins, and then centrifuged ( 1000 rev/min for 5 minL The amount of protocatechuic acid glucoside in the extract was determined by measuring the optical density of 0.2 ml of the supernatant in 2.8 ml of distilled water at 247 nm against a solution of ammonium sulphate of similar concentration. One glucoside unit corresponds to a change in optical density of 0.001 at 247 nm. The left colleterial gland from newly-ecdysed adults and from females 6 days

after decapitation, contain 50 and 80 glucoside units respectively.

For the measurement of the ju'~enile hormone concen- tration sixth instar larvae of different ages were joined to decapitated adult females 4-8 hr after emerging (for technique see BODENSTEIN. 1953). The concentration ~s expressed in glucoside units measured in the left colleterial gland on the sixth day following parabiosis. Ecdysone extraction and the measurement of its concentration was done according to KARLSON and SHAAYA, 1964.

RESULTS

The accumula t i on of g lucos ide in decap i t a t ed f e m a l e s fo l lowing the in jec t ion of d i f ferent a m o u n t s of juven i le h o r m o n e is s h o w n in Fig. 1. F o r comp- a r i son , the in jec ted a m o u n t s of h o r m o n e are a lso g iven in Tenebrio Uni t s (TU). Inc reas ing the a m o u n t of juven i le h o r m o n e is s h o w n in Fig. I. Fo r com- par i son , the i n j ec t ed a m o u n t s of h o r m o n e are a lso g lucos ide was f o u n d to be the same w h e t h e r the individual rece ived 500 ng or 2000 ng h o r m o n e . Thus , maximal syn the t i c abi l i ty of the g land was ob ta ined by in jec t ing 5 0 0 n g ho rmone . The fac t tha t an a m o u n t of on ly 10 ng of the h o r m o n e was needed for the a c c u m u l a t i o n of as m u c h as 150 g lucos ide units , equ iva len t to 15 (TU), suggests this to be a highly sens i t ive b ioassay for juven i le h o r m o n e . The g lucos ide con ten t of the g land in 6 day-o ld f ema les w a s abou t 900 g lucos ide uni ts (BODENSTEIN and SHAAYA. 1968l. T h u s an in jec t ion of 300 ng of the h o r m o n e is a d e q u a t e to cause the accumula t i on of g lucos ide in a m o u n t s c o m p a r a b l e to tha t p roduced normal ly .

Us ing this assay , we s tud ied the va r ia t ion in juve- nile h o r m o n e t i t re in the l a rvae s tage, by parab io t ic fus ion of six ins t a r la rval of d i f ferent ages wi th n e w l y - e c d y s e d decap i t a t ed adul t females . The col- leterial g lands were d i s sec t ed ou t 6 days fo l lowing parab ios i s and the i r g lucos ide c o n t e n t was de ter - mined . This s h o w e d tha t i n the first q u a r t e r of the s tage the re is insuff ic ient j uven i l e h o r m o n e in the la rval pa r t ne r to induce g!ucoside s y n t h e s i s i n the

193

194 EL] SHAAYA

300 --

oo -- ~ J ~ ~ 7 5 0

900 -- ~oo- / _

/I,C) 450 ~ / . - o 500 f.,s ,,.d - soo

/ 300 I -- 150

oc ,.o" - - 7.5 ~I I I I I 2050 oo 2oo 300 400 500

ng of hormone rejected

Fig. I. The effect of different juvenile hormone concen- trations on the synthesis of glucoside. Tenebrio unit (TU) is defined as the mimmum amount of juvenile hormone which is capable of inducing a positive response in 40% of the total pupal insect. O O glucoside unit: O . . . . . O

the activity of the hormone in (TU).

female. The same situation was found in the third and fourth quarters of the,instar (Fig. 2). Glucoside above the expected amount was found in the glands of adults which were parabiosed with larvae in the second quarter of the stage, the maximum being 190 glucoside units.

Determination of ecdysone concentration during the same period revealed that the situation is re- versed (Fig. 2). No ecdysone was found during the first and second quarters of the instar. After this time. however, the ecdysone titre began to rise gradually and reached a maximum of 2.4 (CU)/g equivalent to 35 ng/g insects, shortly before ecdysis. Thus. the secretion of ecdysone occurs when the titre of juvenile hormone is at its lowest level.

DISCUSSION

The general shape of the titre of ecdysone shown in Fig. 2 for Periplaneta americana is remarkably similar to that determined for Bombyx mori (SHAAYA and KARLSON, 1965b). In the penultimate instar of these insects, the titre of hormone begins to increase gradually in the third quarter, resulting in a marked peak shortly prior to ecdysis. It is of interest to note that this peak is 2.4 (CU)and 2 (CU) respectively for Periplaneta and Bombyx. More- over. in both insects the hormone titre drops sharply at ecdysis. The increase in the hormone concentration is correlated to the synthesis of the new cuticle. LANZREIN (1974), showed that in the penultimate instar of the cockroach Nauphoeta cin- erea. the prothoracic gland increases in diameter during the second half of the instar and begins to decrease a few days before ecdysis. This finding ]s consistent with the elevation in ecdysone titre measured in the same period in Periplaneta.

Utilizing the bioassay for juvenile hormone. based on the accumulation of protocatechuic acid glucoside in the left coileterial gland of the cock- roach, it was possible to evaluate the changes in the concentration of juvenile hormone m the larval stage in relation to that of ecdysone. The peak of juvenile hormone occurs in the second quarter of the instar. The secretion of ecdysone follows that of juvenile hormone, and starts when the concentra- tion of the latter is very low.

An unusual finding is that the maximal amount of ecdysone measured in Periplaneta during the instar was 2.4 (CU)/g insect, which is equivalent to about 35 ng of hormone. In the case of juvenile hormone a larva weighing around 500 mg induces the pro- duction of 190 glucoside units, which is equivalent to 15 ng per larva or 30 ng/g insect.

It is commonly thought that juvenile hormone inhibits the changes associated with metamorphis

200~ - - 3 0 @

150 ~ 2.0

I I 30 35 40 ~ ecdysis ecdysis Days ins$or a ~ ~ ins'tar

Fig. 2. Correlation between ecdysone and juvenile hormone titre in the second last instar of Periplaneta. Cailiphora unit (CU) is the amount of ecdysone which is capable of inducing 60% pupation in ligated CaUiphora; a (CU) _ approx 15 ng. O---Juvenile hormone titre expressed in glucoside unit per larva.

O--Ecdysone titre expressed in (CU)/g insect.

Ecdysone and juvenile hormone in Periplaneta 195

(WILLIAMS, 1961; WILLIS, 1969), result ing in the maintenance of larval character . In this communi- cation, it is shown that during larval life, juveni le hormone exer ts its effect on the target cells at the beginning of the instar while ecdysone acts later. Recent ly , studies with Calliphora showed that ecdysone in the absence of juveni le hormone in the last larval instar seems to be responsible for the appearance of giant he terodisperse R N A in the epi- dermal cells (SHAAVA. 1976a, b, c). Part of this R N A species is considered to be the precursor of mes- senger R N A (MELLI and PEMBERTON. 1972L This change in R N A metabol ism is a prerequisi te in the process of t ransformat ion f rom larva to pupa, and it takes place in the absence of juveni le hormone.

R E F E R E N C E S

BODENSTEIN D. (1953) Studies on the humoral mechanism in growth and metamorphosis of the cockroach. Peri- planeta americana. I. Transplantation of integumental structures and experimental parabiosis. J. exp Zool. 123, 189-232.

BODENSTEI N D. and SHAAYA E. (1968) The function of the accessory sex glands in Periplaneta americana (L) I. A quantitative bioassay for the juvenile hormone. Proc. Nat. Acad. Sci. U.S.A. 59, 1223-1230.

BOLLENBACHER W. E., VEDECKIS W. V. and GILBERT L. 1. (1975~ Ecdysone titers and prothoracic gland activity during the larval-pupal development of Manduca sexta. Devl. Biol. 44, 46-53.

KARLSON P. and SHAAYA E. (1964) Der Ecdysontiter wiihrend der Insekten entwicklung. I. Eine Methode zur

Bestimmung des Ecdysongehalts. Y. Insect Physiol. 10, 797-804.

LANZREIN B. (1974) Programming, induction or preven- tion of the breakdown of the prothoracic gland in the cockroach Nauphoeta cinerea. Z Insect Physiol. 21, 367-389.

MELLI M. and PEMaERTON R. E. (1972) New method of studying the precursor-product relationship between high molecular weight RNA and messenger RNA. Nature. New Biol. :?,36, 172-174.

SHAAYA E. (1976a) Separation of high molecular weight hRNA and rRNA by polyacrylamide gel electrophore- sis. Analyt. Biochem. 75, 325-328.

SHAAYA E. ( 1976bl Synthesis and characterization of giant RNA in the epidermal cells of CaUiphora erythro- cephala. Insect Biochem. 6, 553-559.

SHAAYA E. (1976C) Giant RNA in insects 1. Differential changes during Calliphora development and the role of ecdysterone. Biochem. Biophys. Acta 447, 395-405.

SHAAYA E. and KARLSON P. (1965a) Der Ecdysontiter w~ihrend der lnsektenentwicklung, II. Die postembryo- nal EntwicHung der Schmeissfliege CaUiphora erythro- cephala. .L Insect Physiol. 11, 65-69.

SHAAYA E. and KARLSON P. (1965b) Der Ecdysontiter w~ihrend der lnsektenentwicklung, IV. Die Entwicklung der Lepidopteran Bombyx mori L. and Cerura vinula L. Devl. Biol. I I , 424-432.

WILLIAMS C. M. (1961) The juvenile hormone II. Its role in the endocrine control of molting, pupation and adult development in the Cercopia silkworm. Biol. Bull., Woods Hole 121, 572-585.

WILLIS J. H. (1969) The programming of the differentiation and its control by juvenile hormone in Saturniids. J. Embryol. exp. Morph. 22, 27--44.

WILLIS J. H. and BRUNET P. C. J. ~ 1966) Hormonal control of colleterial gland secretion. J. exp. Biol. 44, 363-378.