Prothoracic glands from diapausing pupae of the tobacco hornworm, Manduca sexta, synthesize markedly less ecdysone in vitro in response to prothoracicotropic hormone (PTTH) than do glands from non-diapausing pupae. Impaired steroidogenesis is also observed in glands from diapausing animals exposed to agents that enhance ecdysone synthesis in non-diapausing pupal glands by increasing intracellular levels of cAMP (l-methyl-3-isobutylxanthine, dibutyryl cAMP, and the calcium ionophore A23187). In contrast, prothoracic glands from diapausing pupae synthesize significantly more cAMP in response to PTTH and A23187 than do those from non-diapausing pupae. These observations indicate that the PTTH-refractoriness characteristic of prothoracic glands during diapause results from a lesion in the steroidogenic pathway occurring beyond the level of the PTTH receptor-adenylate cyclase system. The diapause condition of the prothoracic glands (reduced ecdysone synthesis accompanied by enhanced cAMP formation) can be mimicked by extirpation of the brain of a non-diapausing pupa. Thus, cellular changes in the prothoracic glands associated with diapause may arise as a result of the absence of some factor produced by the pupal brain (e.g. PTTH).

When reared under a short-day photoperiod, larvae of the tobacco hornworm, Manduca sexta, enter diapause shortly after pupation. Recent evidence indicates that the neuroendocrine system plays a primary role in regulating the onset of pupal diapause in this insect (Bowen, Bollenbacher & Gilbert, 1984a; Bowen, Saunders, Bollenbacher & Gilbert, 1984b). Following exposure to diapause-inducing conditions during larval development, the release of prothoracicotropic hormone (PTTH) from the pupal brain-retrocerebral complex is suppressed. In the absence of circulating PTTH, haemolymph ecdysteroid titres remain below the threshold required to initiate adult development. In addition to altered PTTH release, the target organs for the neuropeptide, the prothoracic glands, are functionally impaired during diapause. Although the glands of diapausing pupae synthesize low basal levels of the steroid prohormone, ecdysone, they became increasingly refractory to stimulation by PTTH in vitro as diapause progresses (Bowen et al. 1984a). Thus, while the onset of diapause appears to result from events occurring in the larval brain-retrocerebral complex, changes in the prothoracic glands may contribute to the maintenance of chronically reduced ecdysteroid levels.

In the present study, the phenomenon of prothoracic gland refractoriness and its relationship to diapause are explored. Specifically, the following questions are addressed. (1) Is steroidogenic refractoriness a result of atrophy of the glands, i.e. a manifestation of a general reduction in synthetic processes, or alternatively, is ecdysone synthesis altered at specific cellular locations? (2) Does refractoriness arise as an autonomous response of the prothoracic glands to environmental cues, or, as recent data suggest (M. F. Bowen, W. E. Bollenbacher & L. I. Gilbert, unpublished), is it a consequence of a reduction in the activity of the neuroendocrine system? Recent studies of the cellular basis of PTTH-stimulated ecdysone synthesis in non-diapausing pupae indicate that the neurohormone acts to stimulate cyclic AMP (cAMP) synthesis in a calcium-dependent manner, with cAMP in turn enhancing the rate of ecdysone synthesis (Smith, Gilbert & Bollenbacher, 1984, 1985). Using this information, the present study probes the cellular basis of PTTH-refractoriness in the prothoracic glands of diapausing pupae.

Animals

Eggs from a strain of Manduca with a high incidence of diapause were supplied by Dr James Buckner (USDA, Fargo, ND, U.S.A.). Diapausing pupae were obtained by rearing larvae under a short-day photoperiod (LD 12:12) at 25 °C on an artificial diet (Bell & Joachim, 1976). Under this regime, approximately 60% of the pupae entered diapause. Non-diapausing pupae were obtained from our 16-year-old colony, and were reared identically with the exception of photoperiod (LD 16:8). None of the long-day-reared animals entered diapause. Ten-day-old diapausing pupae (D10) were selected as the experimental group as: (a) diapausing pupae at this time are easily distinguished from short-day-reared, non-diapausing cohorts by the absence of pharate-adult characters, such as dissociated fat body in the haemolymph, and eye and wing pad development, and (b) while reduced responsiveness to PTTH can be observed as early as the second day of diapause, refractoriness is maximal in glands from Dio animals (Bowen et al. 1984a). Newly-emerged, non-diapausing pupae (ND0) were chosen as controls, as they represent a stage prior to the initiation of a metamorphic increase in ecdysone synthesis. That is, they comprise a non-diapausing pupal stage analogous to the pre-active condition of the diapausing animals. Once enhanced ecdysone synthesis is initiated, the prothoracic glands undergo pharate-adult development changes (including the onset of cell death) that render them inappropriate as pupal controls.

Brainless pupae were obtained by placing ND0 animals on ice, removing a small square of cuticle from the dorsum of the head, and extirpating the brain. The cuticle was replaced and the edges sealed with melted Tackiwax (Cenco, Chicago, IL). Such pupae remain alive, in a state of arrested development, for several months.

Chemicals, radionuclides and antibody

The calcium ionophore A23187 was purchased from Calbiochem (San Diego, CA). A23187 was prepared as a stock solution in dimethylsulphoxide (Fisher, Pittsburgh, PA) and diluted in Grace’s culture medium (GIBCO, Grand Island, NY) just prior to use. Dibutyryl cyclic AMP (dbcAMP) and l-methyl-3-isobutyl-xanthine (MIX) (Sigma, St Louis, MO) stock solutions were prepared in Grace’s culture medium. [3H] Ecdysone (specific activity 60 Ci mmol−1) and [3H] adenine (specific activity 29 Ci mmol−1) were obtained from New England Nuclear Corp. (Boston, MA) and ICN (Irvine, CA), respectively. The ecdysone antibody used was produced in rabbits against an ecdysone-22-succinyl thryoglobulin synthesized by Dr D. H. S. Horn (C.S.I.R.O., Canberra, Australia) and characterized as described previously (Gilbert, Goodman & Bollenbacher, 1977). All other reagents were obtained from Sigma.

PTTH

Of the two prothoracicotropic peptides found in the pupal brain of Manduca sexta (Mr= 7000 and 28 500; Bollenbacher et al. 1984), the larger form was used in this study. The neurohormone was purified approximately 200-fold by gel-filtration and ion-exchange chromatography as previously described (Bollenbacher et al. 1984). One unit (U) of PTTH, as defined by Bollenbacher, O’Brien, Katahiri & Gilbert (1983), is the amount of prothoracicotropic activity present in a day-1 pupal brain. This quantity is functionally equivalent to the amount of hormone required to half-maximally stimulate 16·7 ND0 pupal prothoracic glands using the in vitro assay described below.

In vitro assay

Pairs of prothoracic glands were dissected in lepidopteran saline and maintained for up to 1 h in Grace’s medium prior to use. Assays were conducted using randomly distributed individual glands, each gland being placed in 25 μl of Grace’s medium containing the experimental or control treatment. Incubations were carried out at 25 °C for the times indicated, and duplicate 10-μ1 samples of the culture medium were assayed for ecdysone directly by radioimmunoassay (RIA). Details of the dissection, incubation, RIA procedures and data analysis by computer were the same as those described previously (Bollenbacher et al. 1983; Warren, Smith & Gilbert, 1984). None of the agents employed (MIX, dbcAMP, PTTH, A23187) interfered with the RIA.

Measurement of cAMP

The accumulation of cAMP in prothoracic glands was assayed by the method of Shimizu, Daly & Creveling (1969), as modified by Meeker & Harden (1982). Glands were pre-incubated individually in 25 μlof Grace’s medium containing 1 μCi [3H]adenine at 25°C for 90 min, rinsed in fresh Grace’s medium, and placed in 25μl of medium containing PTTH or other agents of interest. Following incubation at 25°C for 20 min, a period of time previously found to coincide with enhanced levels of both [3H]cAMP and steroidogenesis (Smith et al. 1984), glands were placed in 200μl ice-cold 25% trichloroacetic acid (TCA) and maintained at 4°C overnight. Chromatographic separation of [3H]cAMP from [3H]ATP in the TCA extract was accomplished by the method of Salomon, Londos & Rodbell (1974), as described previously (Smith et al. 1984). Accumulation of cAMP in the prothoracic glands was expressed as a percentage of conversion of [3H]ATP to [3H]cAMP or {[3H]cAMP/[3H]ATP+[3H]cAMP} × 100. Results obtained with this method correspond to those obtained with a cAMP RIA (Smith et al. 1984).

Ecdysone synthesis by ND0 and D10 prothoracic glands

Previous investigations indicated that prothoracic glands removed from 10-day-old diapausing pupae (D10) show an almost total absence of response in vitro to a single, maximal, dose of pupal brain (PTTH) extract (Bowen et al. 1984a). In the present study, the responses of glands from D10 animals to a range of doses of partially purified big PTTH were examined. Following a 2-h period of incubation, D10 prothoracic glands were essentially unresponsive to all doses of PTTH employed (Fig. 1A). Although basal levels of ecdysone synthesis were slightly, but significantly, reduced in the D10 glands, as compared to non-diapausing day 0 (ND0) controls (P< 0·02, Student’s i-test), this difference was not sufficient to account for the reduced level of synthesis observed in response to PTTH (increased stimulation at 0TUPTTH: D10, × 1·28; ND0, ×4·24). The protein contents of prothoracic glands of D10 and ND0 pupae were not statistically different (ND0 = 21 ± 1 μg gland−1, D10 = 18 ± 2μggland−1). Reduced responsiveness of the D10 glands appeared to result from a reduction in the maximal levels of ecdysone synthesis, rather than a reduced sensitivity to PTTH which would have shifted the doseresponse curve to the right. It is unlikely that the D10 glands would have produced much more ecdysone if the period of incubation had been extended, as PTTH-stimulated ecdysone synthesis is markedly lower in prothoracic glands from diapausing pupae than in glands from ND0 controls for at least 6 h (Bowen et al. 1984a).

Fig. 1.

In vitro ecdysone synthesis by D10 (filled circles) and ND0 (open circles) prothoracic glands, in response to: (A) partially purified big PTTH, (B) dibutyryl cAMP (dbcAMP), (C) l-methyl-3-isobutylxainthine (MIX), (D) calcium ionophore (A23187). Glands were challenged with indicated concentrations of each agent for 2h. Each point represents the mean ± S.E.M. of 10–12 glands.

Fig. 1.

In vitro ecdysone synthesis by D10 (filled circles) and ND0 (open circles) prothoracic glands, in response to: (A) partially purified big PTTH, (B) dibutyryl cAMP (dbcAMP), (C) l-methyl-3-isobutylxainthine (MIX), (D) calcium ionophore (A23187). Glands were challenged with indicated concentrations of each agent for 2h. Each point represents the mean ± S.E.M. of 10–12 glands.

Knowing that cAMP and calcium are involved in the response of the prothoracic glands to PTTH (Smith et al. 1984, 1985), the following agents were examined for their effects on steroidogenesis in glands from diapausing and non-diapausing pupae: (1) the cAMP analogue, dbcAMP; (2) MIX, a potent phosphodiesterase inhibitor; and (3) the calcium ionophore A23187. Each of these agents stimulates ecdysone synthesis up to five-fold in ND0 prothoracic glands, and, although acting at different cellular sites, exerts its steroidogenic effect by increasing intracellular levels of cAMP (Smith et al. 1984, 1985).

As shown in Fig. 1B-D, D10 prothoracic glands exhibited a reduced response to ach agent. In every case, the reduction in ecdysone synthesis as compared to ND0 controls occurred as a result of an impaired maximal response. These findings indicate that the loss of sensitivity to PTTH observed in the prothoracic glands of diapausing pupae may result, at least in part, from alterations in hormone-stimulated ecdysone synthesis beyond the level of cAMP formation. To determine whether such alterations indicated a general reduction in synthetic activity by the glands or a specific steroidogenic lesion, the ability of PTTH to stimulate cAMP formation was assessed.

Accumulation of cAMP by D10 and ND0 prothoracic glands

We tested whether cAMP formation was different in D10 and ND0 prothoracic glands. Since pupal prothoracic glands accumulate little cAMP in the absence of a phosphodiesterase inhibitor (Smith et al. 1984), glands were incubated in the presence of 5 × 10−5 mol l−1 MIX (Fig. 2A). The data reveal that low basal levels of cAMP accumulation were slightly but significantly enhanced (P<0·01) by MIX alone, approximately three-fold in both D10 and ND0 prothoracic glands (Fig. 2A). In ND0 glands, the addition of MIX plus 0-1 UPTTH, a dose previously shown to elicit maximal stimulation of ecdysone synthesis, led to an approximately 10-fold stimulation of cAMP accumulation as compared to MIX alone. This level of cAMP accumulation was similar to that seen in previous experiments with non-diapausing pupal prothoracic glands (Smith et al. 1985). Surprisingly, glands removed from D10 pupae accumulated significantly more cAMP in response to PTTH (17-fold over MIX alone) than did those from non-diapausing pupae (P<0·01). Similar results were obtained using MIX plus the calcium ionophore, A23187, which was previously shown to stimulate cAMP formation in prothoracic glands from non-diapausing pupae at levels similar to those elicited by PTTH (Smith et al. 1985). The addition of A23187 led to an enhancement of cAMP formation in D10 prothoracic glands that was significantly greater than that observed in ND0 glands (P < 0·01).

Fig. 2.

In vitro accumulation of cAMP by prothoracic glands during a 20-min exposure to: Grace’s medium (Basal); 5× l0−5moll−1 l-methyl-3-isobutylxanthine (MIX); 0·1 U PTTH+MIX; or 5×10−5moll−1 A23187+MIX. (A) Responses of glands from ND0 pupae (white bars) vs D10 pupae (stippled bars). (B) Responses of glands from ND0pupae (open bars) vs ND0 pupae 2 days (BrX2, cross-hatched bars) or 10 days (BrX10, filled bars) after brain removal. Bars represent mean ±S.E.M.; sample size indicated above bars.

Fig. 2.

In vitro accumulation of cAMP by prothoracic glands during a 20-min exposure to: Grace’s medium (Basal); 5× l0−5moll−1 l-methyl-3-isobutylxanthine (MIX); 0·1 U PTTH+MIX; or 5×10−5moll−1 A23187+MIX. (A) Responses of glands from ND0 pupae (white bars) vs D10 pupae (stippled bars). (B) Responses of glands from ND0pupae (open bars) vs ND0 pupae 2 days (BrX2, cross-hatched bars) or 10 days (BrX10, filled bars) after brain removal. Bars represent mean ±S.E.M.; sample size indicated above bars.

Ecdysone synthesis during the same 20-min period is shown in Table 1. Steroidogenesis was clearly reduced in the D10 glands, corroborating the results shown in Fig. 1. In the ND0 glands, similar levels of ecdysone synthesis were elicited by MIX, PTTH+MIX, or A23187+MIX, despite markedly different levels of cAMP accumulation. This result indicates that while cAMP synthesis is likely to play an important role in pupal steroidogenesis, the dramatic accumulation of cAMP obtained with PTTH or ionophore in the presence of MIX is not essential for glandular activation. Such an observation is in keeping with the ability of PTTH alone to stimulate ecdysone synthesis in ND0 glands in the absence of a detectable increase in cAMP accumulation (Smith et al. 1984).

Table 1.

Ecdysone synthesis by prothoracic glands from diapausing and non-diapausing pupae concomitant with cyclic AMP production shown in Fig. 2A 

Ecdysone synthesis by prothoracic glands from diapausing and non-diapausing pupae concomitant with cyclic AMP production shown in Fig. 2A
Ecdysone synthesis by prothoracic glands from diapausing and non-diapausing pupae concomitant with cyclic AMP production shown in Fig. 2A

Of particular relevance to the present study, the results shown in Fig. 2 indicate that prothoracic glands from diapausing pupae are indeed capable of synthesizing cAMP in response to both receptor-mediated (PTTH) and non-receptor-mediated (A23187) secretagogues. Thus, refractoriness is unlikely to be a consequence of a general reduction in anabolic activity. Further, the glands from diapausing animals appear to be hypersensitive to both PTTH and ionophore as far as cAMP formation is concerned.

Effects of brain removal on prothoracic gland function

The above data indicated that the refractoriness observed during diapause arose from an inability of the prothoracic glands to synthesize ecdysone in response to increased intracellular levels of cAMP. Of equal interest, however, was the relationship of this condition to the principal cause of pupal diapause in Manduca, the photoperiodically curtailed release of PTTH (Bowen et al. 1983, 1984). Conceivably, refractoriness of the prothoracic glands during diapause could be a consequence of the absence of PTTH release itself, and/or the absence of another cerebral neuropeptide necessary for sustained responsivity of the glands after pupation. To investigate these possibilities, brains were removed from non-diapausing day-0 pupae and the synthetic events in the prothoracic glands examined.

The ability of prothoracic glands from brainless pupae to synthesize ecdysone is illustrated in Fig. 3. When challenged with PTTH (0·1 U) 2 or 10 days after brain removal, these prothoracic glands showed a significant (P<0·01) reduction in ecdysone synthesis. In addition, prothoracic glands from brainless pupae were significantly less responsive to a l0mmoll−1 dose of dbcAMP (P<0·01). Further, intracellular levels of cAMP following a 20-min exposure to PTTH+MIX were enhanced significantly in prothoracic glands from brainless pupae, as compared to intact ND0 controls (P< 0·01, Fig. 2B). These results indicate that, as in the case of prothoracic glands from diapausing pupae, reduced responsiveness to PTTH following brain removal occurs at a point beyond cAMP formation. The similarity at the cellular level between prothoracic glands removed from diapausing and debrained pupae strongly suggests that the refractory states of the two groups are functionally similar, and that the condition of the glands from diapausing pupae results from the absence of some factor originating in the pupal brain.

Fig. 3.

In vitro ecdysone synthesis by glands from ND0 pupae (open bars), from ND0 pupae 2 days (cross-hatched bars) or 10 days (filled bars) after brain removal. Glands were challenged for 2 h with Grace’s medium (Basal), PTTH (0·1 U) or dibutyryl cAMP (10 mmol l−1 ). Bars represent mean ±S.E.M.; samples sizes indicated above bars.

Fig. 3.

In vitro ecdysone synthesis by glands from ND0 pupae (open bars), from ND0 pupae 2 days (cross-hatched bars) or 10 days (filled bars) after brain removal. Glands were challenged for 2 h with Grace’s medium (Basal), PTTH (0·1 U) or dibutyryl cAMP (10 mmol l−1 ). Bars represent mean ±S.E.M.; samples sizes indicated above bars.

The present results indicate that prothoracic glands removed from Dio pupae of Manduca sexta synthesize less ecdysone in response to PTTH than do glands from ND0 animals. Prothoracic glands from diapausing pupae are similarly unresponsive to agents that act by increasing intracellular levels of cAMP (MIX, dbcAMP and A23187). Paradoxically, D10 prothoracic glands synthesize more cAMP than ND0 glands in response to PTTH and A23187. These data argue strongly for a postreceptor lesion in the prothoracic glands as the basis for cellular refractoriness to PTTH during diapause.

Removal of the brain mimics the diapause condition of the prothoracic glands with regard to both ecdysone synthesis and cAMP formation. Thus, the present study corroborates and strengthens the suggestion that changes in the prothoracic glands associated with diapause may be a consequence of the absence of an agent or agents produced by the pupal brain (Bowen et al. 1984a, and unpublished information). The possibility exists that PTTH itself may be the responsible factor. In rats, removal of adrenocorticotropin (ACTH) through hypophysectomy leads to adrenal atrophy, preceded or accompanied by reduced corticosteroid synthesis in response to ACTH (Grahame-Smith, Butcher, Ney & Sutherland, 1967; Sayers & Beall, 1972; Holmes, Neto & Field, 1980). Associated with a reduction in corticosteroid production, there is an increase in ACTH-stimulated cAMP accumulation (Sayers & Beall, 1972; Holmes et al. 1980) and in [125I]ACTH binding to the adrenal glands (Golder & Boyns, 1972), suggesting an increase in either the affinity or number of ACTH receptors. Similar changes have been observed in the rat thyroid following removal of thyrotropin (TSH) via hypophysectomy (Zakarija & McKenzie, 1977; Friedman, Land, Levasseur & Burke, 1979, Holmes & Field, 1980). Reduced responsiveness to TSH may result from a decrease in cAMP-dependent protein kinase activity, as indicated by an approximately 50 % decrease in the activity of this enzyme in hypophysectomized animals (Friedman et al. 1979; Combest & Russell, 1983). Studies on mammalian systems suggest that peptides which exert a trophic effect on their target tissues (maintenance of tissue weight and hormonal responsiveness) appear to do so via maintenance of enzyme activity beyond adenylate cyclase. In the absence of the trophic peptide, the activity of such enzymes is reduced, with a concomitant increase in peptide-stimulated second messenger production.

The present data are in remarkable accordance with the above findings for mammalian endocrine systems. We suggest that an absence of low (trophic) levels of PTTH during the first few days of pupal life, as a result of either larval exposure to short-day photoperiod (Bowen et al. 1984a,b) or brain extirpation, leads to decreased steroidogenic capacity accompanied by enhanced cAMP formation. The cellular basis of refractoriness to PTTH by the prothoracic glands occurs beyond cAMP formation, possibly at the level of cAMP-dependent protein kinase activity. Initial studies of protein kinase activity in the prothoracic glands indicate a 30–40 % reduction in the cellular levels of cAMP-dependent protein kinase activity in glands from D10 as compared to ND0 pupae (W. A. Smith, W. L. Combest & L. I. Gilbert, unpublished observation). However, although the results of the present study suggest a trophic role for PTTH in the regulation of prothoracic gland function, confirmation of this will require further experimentation. In particular, it will be important to determine whether administration of PTTH to day-0, de-brained pupae via brain implantation or infusion of purified hormone is capable of preventing a subsequent loss in steroidogenic capacity.

The cellular basis of increased cAMP accumulation by the prothoracic glands of diapausing animals is also conjectural at present. The presence of MIX during the incubation of both D10 and ND0 glands argues against reduced phosphodiesterase activity in the D10 group as the cause of enhanced levels of cAMP. It thus appears that cAMP synthesis is altered during diapause. Such a change may occur through an increase in the number of PTTH receptors or their affinity for the neurohormone, through enhanced interaction of the PTTH receptor with the catalytic unit of adenylate cyclase, or through an increase in the activity of the catalytic unit of the enzyme. The ability of the calcium ionophore to generate cAMP levels in diapausing glands equivalent to those generated by PTTH suggests that a change in the receptor cannot explain completely the enhanced capacity for cAMP production during diapause. This leaves the regulatory and/or catalytic unit of the adenylate cyclase as likely sites at which cAMP synthesis may be altered.

Since the prothoracic glands are refractory during diapause, the question arises as to how this condition is normally reversed to permit the resumption of development. Environmental cues, most probably an increase in temperature, must in some manner stimulate an increase in ecdysone synthesis, either by affecting the prothoracic glands directly or by stimulating the production of trophic factors (e.g. PTTH) that ultimately stimulate steroidogenesis. In the bollworm, Heliothis, PTTH is released in diapausing animals; however, the prothoracic glands remain inactive at low temperatures and become active as the temperature increases (Heliothis zea, Meola & Adkisson, 1977; Heliothis punctiger, Browning, 1981). Recent evidence indicates that, rather than responding directly to increased temperature, the glands are activated by a temperature-sensitive increase in levels of a humoral factor required for ecdysone synthesis (Meola & Gray, 1984). Similarly, in most insect species, a direct influence of environmental cues on prothoracic gland function has not been clearly demonstrated. However, activation of the prothoracic glands of diapausing pupae in a manner independent of the brain, particularly in response to temperature change and wounding, is well-documented (Papilio, Numata & Hidaka, 1984; Antheraea, McDaniel & Berry, 1967; McDaniel, Johnson, Saun & Berry, 1976; Manduca, Judy, 1972).

At present, the favoured mechanism for the restoration of prothoracic gland activity in Manduca is the resumption of PTTH release. Implantation of brains into brainless, non-diapausing pupae does re-establish synthetic competence of the prothoracic glands by the ninth day after implantation (M. F. Bowen, W. E. Bollenbacher & L. I. Gilbert, unpublished observations). Because the glands remain capable of producing cAMP in response to PTTH, the possibility exists that a chronic enhancement of intracellular levels of cAMP, as opposed to an acute increase, may be the mechanism by which synthetic capacity is eventually restored. In the rat adrenal glands, refractoriness arising from hypophysectomy can be reversed, at least in part, by prolonged (5-day) administration of dbcAMP (Ney, 1969). An additional mechanism by which ecdysone synthesis may be re-initiated is through the action of an agent that does not act via cAMP. One such agent, produced by the Manduca fat body in response to juvenile hormone (Gruetzmacher, Gilbert & Bollenbacher, 1984a; Gruetzmacher et al. 1984b) and apparently identical to an agent found in the haemolymph of developing Manduca larvae (Watson et al. 1985), stimulates ecdysone synthesis in both larval and non-diapausing pupal prothoracic glands without enhancing cAMP formation (Smith, Watson, Gilbert & Bollenbacher, 1986). This compound, a 30 000 Da protein, is currently hypothesized to act as a carrier molecule for, or to enhance the transport of, a sterol substrate necessary for ecdysone synthesis (Watson et al. 1985). Although the cellular basis for the termination of pupal diapause is not known with certainty, the current study denotes the sites at which environmental or systemic cues may restore prothoracic gland function.

We wish to thank Ms Susan Whitfield and Ms Shelia King for their assistance in preparing the manuscript. This research was supported by the following grants: NIH AM-30118 and NSF DCB-8502194 to LIG, NIH NS-18791 and AM-31642 to WEB, and NIH National Research Service Awards AM-07272 to WAS and AM-06855 to MFB.

Bell
,
R. A.
&
Joachim
,
F. G.
(
1976
).
Techniques for rearing laboratory colonies of tobacco hornworms and pink bollworms
.
Ann. ent. Soc. Am
.
69
,
365
373
.
Bollenbacher
,
W. E.
,
Katahira
,
E. J.
,
O’brien
,
M. A.
,
Gilbert
,
L. I.
,
Thomas
,
M. K.
,
Agui
,
N.
&
Baumhover
,
A. H.
(
1984
).
Insect prothoracicotropic hormone: evidence for two molecular forms
.
Science, N.Y
.
224
,
1243
1245
.
Bollenbacher
,
W. E.
,
O’brien
,
M.
,
Katahira
,
E. J.
&
Gilbert
,
L. I.
(
1983
).
A kinetic analysis of the action of the insect prothoracicotropic hormone
.
Molec. cell. Endocr
.
32
,
27
46
.
Bowen
,
M. F.
,
Bollenbacher
,
W. E.
&
Gilbert
,
L. I.
(
1984a
).
In vitro studies on the role of the brain and prothoracic glands in the pupal diapause of Manduca sexta
.
J. exp. Biol
.
108
,
9
24
.
Bowen
,
M. F.
,
Saunders
,
D. S.
,
Bollenbacher
,
W. E.
&
Gilbert
,
L. I.
(
1984b
).
In vitro reprogramming of the photoperiodic clock in an insect brain-retrocerebral complex
.
Proc. natn. Acad. Sci. U.S.A
.
81
,
5881
5884
.
Browning
,
T. O.
(
1981
).
Ecdysteroids and diapause in pupae of Heliothis punctiger
.
J. Insect Physiol
.
27
,
715
721
.
Combest
,
W. L.
&
Russell
,
D. H.
(
1983
).
Alteration in cyclic AMP-dependent protein kinases and polyamine biosynthetic enzymes during hypertrophy and hyperplasia of the thyroid in the rat
.
Molec. Pharmac
.
23
,
641
647
.
Friedman
,
Y.
,
Land
,
M.
,
Levasseur
,
S.
&
Burke
,
G.
(
1979
).
Demonstration of a tonic regulatory thyrotropin effect on thyroid function
.
Endocrinology
104
,
467
475
.
Gilbert
,
L. L
,
Goodman
,
W.
&
Bollenbacher
,
W. E.
(
1977
).
Biochemistry of regulatory lipids and sterols in insects
.
In International Review of Biochemistry, Biochemistry of Lipids II
, Vol.
14
, (ed.
T. W.
Goodwin
), pp.
1
50
.
Baltimore
:
University Park Press
.
Golder
,
M. P.
&
Boyns
,
A. R.
(
1972
).
Selective uptake of radioactivity by the adrenal cortex of dexamethasone-treated guinea pigs after the administration of 131l-labelled a1−24-adrenocorticortrophin
.
J. Endocrin
.
53
,
277
287
.
Grahame-Smith
,
D. G.
,
Butcher
,
R. W.
,
Ney
,
R. L.
&
Sutherland
,
E. W.
(
1967
).
Adenosine 3′,5′-monophospate as the intracellular mediator of the action of adrenocorticotropic hormone on the adrenal cortex
.
J, biol. Chem
.
242
,
5535
5541
.
Gruetzmacher
,
M. C.
,
Gilbert
,
L. I.
&
Bollenbacher
,
W. E.
(
1984a
).
Indirect stimulation of the prothoracic glands of Manduca sexta by juvenile hormone: evidence for a fat body stimulatory factor
.
J. Insect. Physiol
.
30
,
771
778
.
Gruetzmacher
,
M. C.
,
Gilbert
,
L. L
,
Granger
,
N. A.
,
Goodman
,
W.
&
Bollenbacher
,
W. E.
(
1984b
).
The effect of juvenile hormone on prothoracic gland function during the larvalpupal development of Manduca sexta: an in situ and in vitro analysis
.
J. Insect Physiol
.
30
,
331
340
.
Holmes
,
S. D.
&
Field
,
J. B.
(
1980
).
Effects of endogenous TSH on the in vitro responsiveness and binding of TSH in rat thyroid tissue
.
Endocrine Res. Commun
.
7
,
205
209
.
Holmes
,
S. D.
,
Neto
,
F. R.
&
Field
,
J. B.
(
1980
).
Effect of in vivo treatment with adrenocorticotropin and hypophysectomy on adrenal adenosine 3′,5′-monophosphate and corticosterone responsiveness to adrenocorticotropin stimulation in vitro
.
Endocrinology
107
,
432
437
.
Judy
,
K. J.
(
1972
).
Diapause termination and metamorphosis in brainless tobacco hornworms (Lepidoptera)
.
Life Sci
.
11
,
605
611
.
Mcdaniel
,
C. N.
&
Berry
,
S. J.
(
1967
).
Activation of the prothoracic glands of Antheraea polyphemus
.
Nature, Lond
.
214
,
1032
1033
.
Mcdaniel
,
C. N.
,
Johnson
,
E.
,
Saun
,
T.
&
Berry
,
S. J.
(
1976
).
Ultrastructure of active and inhibited prothoracic glands
.
J. Insect Physiol
.
22
,
473
481
.
Meeker
,
R. B.
&
Harden
,
T. K.
(
1982
).
Muscarinic cholinergic receptor-mediated activation of phosphodiesterase
.
Molec. Pharmac
.
22
,
3110
3119
.
Meóla
,
R. W.
&
Adkisson
,
P. L.
(
1977
).
Release of prothoracicotropic hormone and potentiation of developmental ability during diapause in the bollworm, Heliothis zea
.
J. Insect Physiol
.
23
,
683
688
.
Meóla
,
R. W.
&
Gray
,
R.
(
1984
).
Temperature-sensitive mechanism regulating diapause in Heliothis zea
.
J. Insect Physiol
.
30
,
743
749
.
Ney
,
R. L.
(
1969
).
Effects of dibutyryl cyclic AMP on adrenal growth and steroidogenic capacity
.
Endocrinology
84
,
168
170
.
Numata
,
H.
&
Hidaka
,
T.
(
1984
).
Role of the brain in post-diapause adult development in the swallowtail, Papilio xuthus
.
J. Insect Physiol
.
30
,
165
168
.
Salomon
,
Y.
,
Londos
,
C.
&
Rodbell
,
M.
(
1974
).
A highly sensitive adenylate cyclase assay
.
Analys. Biochem
.
58
,
541
548
.
Sayers
,
G.
&
Beall
,
R. J.
(
1972
).
Isolated adrenal cortex cells: hypersensitivity to adrenocorticotropic hormone after hypophysectomy
.
Science, N.Y
.
179
,
1300
1331
.
Shimizu
,
H.
,
Daly
,
J. W.
&
Creveling
,
C. R.
(
1969
).
A radioisotope method for measuring the formation of adenosine 3′,5′-monophosphate in incubated slices of brain
.
J. Neurochem
.
16
,
1609
1619
.
Smith
,
W. A.
,
Gilbert
,
L. I.
&
Bollenbacher
,
W. E.
(
1984
).
The role of cyclic AMP in ecdysone synthesis
.
Molec. cell. Endocr
.
37
,
285
294
.
Smith
,
W. A.
,
Gilbert
,
L. I.
&
Bollenbacher
,
W. E.
(
1985
).
Calcium-cyclic AMP interactions in prothoracicotropic hormone stimulation of ecdysone synthesis
.
Molec. cell. Endocr
.
39
,
71
78
.
Smith
,
W. A.
,
Watson
,
R. D.
,
Gilbert
,
L. I.
&
Bollenbacher
,
W. E.
(
1986
).
The steroidogenic action of haemolymph stimulatory factor in Manduca sexta. Comparisons with prothoracicotropic hormone
.
Insect Biochem. (in press)
.
Warren
,
J. T.
,
Smith
,
W. A.
&
Gilbert
,
L. I.
(
1984
).
Simplification of the ecdysteroid radioimmunoassay with the use of protein A from Staphylococcus aureus
.
Experientia
40
,
393
394
.
Watson
,
R. D.
,
Ciancio
,
M.
,
Gunnar
,
W. P.
,
Gilbert
,
L. I.
&
Bollenbacher
,
W. E.
(
1985
).
Regulation of insect prothoracic glands: existence of a hemolymph stimulatory factor in Manduca sexta
.
J. Insect Physiol
.
31
,
487
494
.
Zakarua
,
M.
&
Mckenzie
,
J. M.
(
1977
).
Effects of thyrotropin and thyroid hormones in vivo on thyroid responsiveness to thyrotropin in vitro
.
Endocrine Res. Commun
.
4
,
343
355
.