GENERAL AND COMPARATIVE ENDOCRINOLOGY 42, 129- 133 (1980) Activity of Juvenile Hormone Acid in Brainless, Allatectomized Diapausing Cecropia Pupae G. BHASKARAN,G. DELEON, B. LOOMAN,P. D. SHIRK,'AND H. ROLLER Institute of Developmental Biologj~,Texas A&M University, College Stution, Texas 77843 Accepted March 31, 1980 Pupal diapause in the cecropia silkmoth is terminated by activation of the prothoracic glands. Implantation of adult male cecropia corpora allata or injection of juvenile hormone-I (JH-I) or juvenile hormone-I acid (JH-I acid) causes initiation of development in debrained, allatectomized diapausing pupae. The subsequent development results in formation of sec- ond pupae or pupal-adult intermediates. The dose-response patterns for JH-I acid and JH-I are nearly identical. These data suggest that JH-I acid activates prothoracic glands and in addition acts like a morphogenetic hormone. Diapause in saturniid pupae results from observations that adult male CA can acti- the programmed inactivation of the brain- vate PG and cause pupal-adult develop- prothoracic gland system and is termin- ment were puzzling since JH acid was re- ated by activation of the prothoracic ported to be inactive when bioassayed glands (PG) which leads to the secretion of (Slade and Zibitt, 1972) and has generally ecdysone (Williams, 1952). Removal of the been considered to be an inactive precursor brain from such pupae keeps them in a state or catabolite with no known hormonal ac- of permanent diapause (dauerpupae) which tivity. To resolve this paradox, we injected can be terminated by implantation of active various concentrations of JH-I acid into brains or active corpora allata (Williams, brainless, allatectomized diapausing ce- 1952, 1959; Ichikawa and Nishiitsutsuji- cropia pupae. We report here that JH-I acid Uwo, 1959). Juvenile hormone (JH), far- does indeed terminate diapause as well as nesol, methyl famesoate, and various other induce formation of second pupae or compounds with JH activity are also effec- pupal- adult intermediates. tive in terminating pupal diapause (Gilbert and Schneiderman. 1959: Krishnakumaran MATERIALS AND METHODS and Schneiderman, 1965). Besides the pre- Diapausing cecropia pupae were purchased from sumed activation of the prothoracic glands, Lepidoptera Company, Chicago, Illinois. Brain and corpora allata-corpora cardiaca (CA-CC) complexes JH and JH analogs also prevent normal were surgically removed (Williams, 1959) from metamorphosis and hence dauerpupae diapausing pupae, chilled at 4-6' for 2-3 months. treated with sufficient JH often develop After surgery, the pupae were kept in plastic contain- into second pupae or pupal-adult inter- ers in a room maintained at 27" and 70-80% relative mediates. humidity with a 16:8 light-dark cycle. None of these animals showed any signs of development at the end of Recently, it was discovered that corpora 1 week and were thereafter considered dauerpupae allata (CA) of adult male cecropia lack an and used for our experiments. The age of the pupae at active JH acid methyltransferase and in the time of experimentation varied from 7 to 90 days vitro secrete virtually no JH but instead se- after brain-CA-CC complex removal. The state of crete JH-I and JH-11 acid (M. Peter, unpub- development of the experimental animals was evaluated 35 days after treatment. lished observation). Therefore, the earlier Racemic JH-1 was obtained from Eco-Control, Cambridge, Massachusetts, and purified by high- ' Present address: Department of Biology, Univer- resolution liquid chromatography. JH-I acid, prepared sity of Oregon, Eugene, Oreg. 97403. by alkaline hydrolysis of JH, was provided by Dr. K. 129 00 16-6480/80/090129-05$01.0010 Copyright @ 1980 by Academic Press, Inc. All rights of reproduction in any form resewed. 130 BHASKARAN ET AL. Dahm. JH-I was diluted in olive oil and the more polar pupal-adult intermediates (Table 2). At JH-I acid in 10% ethanolic Ringer. lower concentrations (1.0-25 pglpupa) JH-I seemed to have more activity but we RESULTS AND DISCUSSION are not certain whether this is a genuine In view of the interesting sexual di- difference or simply due to factors as- morphism in CA activity of adult cecropia sociated with the solvent's dispersal in the we tested whether corpora allata from 1- to animal or to the greater metabolic instabil- 3-day-old adult males and females differ in ity of JH-I acid. The response to JH-I acid their ability to terminate diapause in brain- could not be due to the presence of JH-I as less pupae. The results shown in Table 1 a contaminant, because if such contarnina- reveal that male CA are far more active tion existed it was less than 1% (Dr. K. than female CA. All animals which devel- Dahm, personal communication). Nearly oped after implantation of two pairs of male all the animals which received 100.0 pgl CA were pupal-adult intermediates and pupa of either JH-I or JH-I acid broke three of the five animals with one pair of diapause and a number of them developed implanted male CA metamorphosed into into second pupae. All the control pupae intermediates. In contrast, of those animals remained in diapause and continued to re- which received two pairs of female CA only main so even after 6 months. one initiated development and it metamor- It is unlikely that JH-I acid directly stim- phosed into a normal adult. Such differ- ulated epidermal cells to develop in these ences in prothoracicotropic and mor- dauerpupae because injection of 100 pg of phogenetic activities between male and the acid into isolated abdomens (N = 5) female CA were not reported earlier either produced no response. Isolated abdomens for cecropia (Williams, 1959) or for which received adult male CA or JH-I failed Philosamia (Ichikawa and Nishiitsutsuji- to develop, but injection of 10.0 ~g of P- Uwo, 1959). ecd ysone promoted adult development. Next we injected various dosages of JH-I These results clearly indicate that JH-I acid and JH-I acid ranging from 1.0 to 100.0 pg/ (and JH-I) activated the PG in the brainless, pupa. The results showed that in general allatectomized pupae. However, it should JH-I acid was almost as active as JH-I in be noted that the doses of JH-I and JH-I terminating diapause and many of the re- acid used in the present study are likely to cipients developed into second pupae or be above physiological levels and evidence TABLE 1 ACTIVATIONOF DEVELOPMENTIN BRAINLESS, ALLATECTOMIZED" DIAPAUSING CECROPIA PUPAEBY IMPLANTATIONOF CORPORA ALLATA (CA) Resulting developmental response Implant No. No. No. No. pupal-adult No. Average (pair per host) hostsb developed adults intermediates 2nd pupae scoreC 2 adult male CA 20 10 0 10 0 3.0 1 adult male CA 9 5 2 3 0 2.0 2 adult female CA 6 1 1 0 0 0.0 Sham implantation 47" 0 0 0 0 - Surgical procedures led also to removal of the corpora cardiaca. * Included males and females. Morphogenetic effects scored according to Williams (1961) on a scale of 0-5, with 0 being a normal adult and 5 a second pupa. "No sign of development was observed even after 6 months. HORMONAL ACTIVITY OF JH-I ACID 131 TABLE 2 EFFECTSOF JUVENILEHORMONE-I AND JUVENILEHORMONE-[ ACID ON TERMINATIONOF DIAPAUSE AND ON ADULTDEVELOPMENT IN BRAINLESS, ALLATECTOMIZED" DIAPAUSING CECROPIA PUPAE Resulting developmental response Treatment No. No. No. No. pupal-adult No. Average (&pupa) hosts" developed adults intermediates 2nd pupae score' JH-I 1.0 JH-I 10.0 JH-I 25.0 JH-I 50.0 JH-I 100.0 JH-I acid 1.0 JH-I acid 10.0 JH-I acid 25.0 JH-I acid 50.0 JH-I acid 100.0 Olive oil 100 p1 10% ethanolic Ringer 100 pl No injection " Surgical procedures led also to removal of the corpora cardiaca. Included males and females. Morphogenetic effects scored according to Williams (1961) on a scale of 0-5, with 0 being a normal adult and 5 a second pupa. "0 sign of development was observed even after 6 months. is lacking that the CA play any role in acti- both the lack of sex specificity in the re- vation of the PG during the early period of sponse to JH-I acid and that the male ASG adult development. are incapable of methylating JH acids until Does JH-I acid directly activate PG or is much later in development (Weirich and it first converted to JH-I? Since the CA Culver, 1979) rule out the ASG as a source were also removed from the pupae, the of JH. Pupal epidermal cells are, however, conversion would have had to occur either capable of methylating JH-I acid in small in the PG itself or in some other tissue and quantities (<0.0 1% of that in the medium in then be transported to the PG. At present vitro; Shirk and Bhaskaran, unpublished we have no evidence to indicate whether observation). This ability may be shared by such a conversion to JH-I is necessary. The other tissues in vivo, including the PG. most efficient conversion in whole animals Hence, we cannot, at present, disregard the observed so far has been in adult male alternative that conversion of the acid to moths which methylate approximately 6% the hormone in the PG itself is the primary of the injected JH-I acid to JH-I (Metzler et event in activation of ecdysone secretion by al., 1972). Adult male accessory sex glands the PG. (ASG) convert less than 6% of JH-I acid to The possibility that JH-I is first con- JH-I in vivo as well as in vitro (Shirk, 1978).