Physiology of Pupal Ecdysis in the Tobacco Hornworm, Manduca Sexta: II. Chemistry, Distribution, and Release of Eclosion Hormone At Pupal Ecdysis

The adult eclosion of large moths from their pupal case is triggered by a peptide hormone, the eclosion hormone (Truman & Riddiford, 1970). The previous paper in this series (Truman, Taghert & Reynolds, 1980) presented evidence that the eclosion hormone might also initiate pupal ecdysis in the tobacco hornworm, Manduca sexta. It was shown that exogenous eclosion hormone can trigger pupal ecdysis behaviour and that a factor with eclosion hormone-like activity appears in the blood of pharate pupae about 30 min before ecdysis. The activity in the blood was sufficient to cause ecdysis when injected into another pharate pupa.

This evidence is sufficient to show that a hormone with eclosion hormone-like properties is involved in pupal ecdysis, but it does not prove that this factor is identical with eclosion hormone. This paper reports on the presence of an eclosion hormone-like factor in the pharate pupal central nervous system (CNS), its distribution within the CNS, the dynamics of its release, and the factor’s biochemical characteristics and activity in a number of different bioassays. We conclude that the hormones controlling pupal ecdysis and adult eclosion are the same.

The rearing and staging of Manduca sexta larvae were as described in the previous report (Truman et al. 1980). Diapausing pupae of Hyalophora cecropia were obtained from dealers. Stimulation of development and the methods of abdomen isolation from pharate adults were as described in Truman (1978).

Tissues were homogenized in distilled water containing a few crystals of phenylthiourea in ground-glass homogenizers, heated to 80 °C for 5 min and centrifuged at 1000 g at room temperature for 20 min. The resultant supernatant was used immediately or stored at − 20 °C for at most a few days before use.

For gel filtration, tissues were treated as above (50 tissues homogenized in 0·9 ml dH₂O) and acidified by addition of 0·1 ml 1·0 N-HAC. Following centrifugation at 17500 g for 20 min at 4 °C, supernatants were chromatographed immediately on Sephadex G-50 fine (Pharmacia). One ml samples were layered on a 1·6x30 cm column, eluted at room temperature with 0·1 N-HAC with a mechanically controlled flow rate of approximately 8 ml/cm²/h. The column was calibrated using blue dextran (Pharmacia), cytochrome C (Sigma), insulin (CalBioChem), bacitracin (Sigma) and KC1.

For electrofocusing the relevant fractions from replicate runs through Sephadex were pooled, lyophylized, and resuspended in 0·1 N-HAC and 0·1% bacitracin (to enhance the stability of the peptides; Mumby, Truman & Reynolds, in preparation) and layered over pre-cast electrofocusing gels (0·5 × 10 cm) containing wide-range ampholines (pH 3−9; Bio-Rad). The gels were run for 22 h at 4 °C at 200 V. Gels were then sliced (3 mm thickness) and the slices eluted for 48 h in 0·1 N-HAC and 0·1 % bacitracin at 4 °C.

Calibrations were performed by eluting slices from a simultaneously run blank gel in freshly boiled dH₂O for 2−3 h at room temperature, then measuring the pH of the eluents.

A number of biological assays were used in the present study. For the pupal ecdysis assay described in the preceding paper (Truman et al. 1980), anterior shrinkage (AS) stage pharate pupae were injected with tissue extracts or aliquots of unbuffered column fractions. Positive scores were calculated as the reciprocal of the latency (min) × 10⁴. As described previously, negative assays were scored as 51.

A second assay utilized abdomens isolated from pharate adult H. cecropia. The responses of the abdomen to injections of various fractions were recorded as described in Truman (1978) and were scored according to the criteria in Mumby, Truman & Reynolds (in preparation). Briefly, these were: (1) a weak, drawn-out pre-eclosion behaviour; (2) a normal pre-eclosion behaviour but no eclosion behaviour; (3) a weak eclosion behaviour; (4) a strong eclosion behaviour with shedding of the pupal cuticle. The assay based on this scoring system is about 10-fold less sensitive than the pupal ecdysis assay and is roughly linear with the log of hormone concentration.

The third assay utilized wings from pharate adult Manduca sexta. Fractions in acid were buffered (Reynolds, 1977) before injection of 10 μ1 into the wing veins. The score (in mm) is based on the difference in plasticity in the test wing compared with a paired control and measured according to the technique of Reynolds (1977) as modified by Reynolds & Truman (1980).

In titering the amount of eclosion hormone activity in various organs, activity was expressed in terms of ‘units’. One unit is equivalent to the amount of activity extracted from a CC-CA pair from a pharate adult.

Some experiments involved removing the brain from animals on the night of wandering (Truman & Riddiford, 1974), i.e. 3−4 days since the last larval ecdysis and 4 days before the larval-pupal ecdysis. Animals were anaesthetized by immersion in water, then suspended by a loose neck clamp over a stream of carbon dioxide. A small rectangle of cuticle was completely removed just lateral to the inverted Y suture of the head. The brain was excised and either discarded or reimplanted to its original position. The excised piece of cuticle was replaced over the hole and sealed in place with melted Tackiwax (Cenco).

The nervous systems were removed from pharate pupae that were approximately 4−8 h before ecdysis. Ganglia from various regions were combined, extracted as described in the Materials and Methods and assayed for their eclosion hormone activity using the pupal ecdysis assay described in the previous paper (Truman et al. 1980). The distribution of eclosion hormone activity is summarized in Table 1. The largest store of the hormone appeared in the abdominal ganglia with lesser fcnounts present in the brain and the thoracic ganglia. The assay detected no hormone in the subesophageal ganglion or the corpora cardiaca-corpora allata (CC-CA) complex. The lack of eclosion hormone activity in this last structure is in striking contrast to the pharate adult stage in which the majority of the cephalic store of eclosion hormone is stored in the CC-CA complex (Truman, 1973).

Pupal ecdysis is accompanied by the abrupt appearance of substantial eclosion hormone activity in the blood (Truman et al. 1980). The results described above showed that eclosion hormone activity is widely distributed in the pharate pupal CNS and consequently there are a number of potential sources of this blood-borne activity. We examined the two major sites of activity, the brain and the abdominal nerve cord, from AS stage pharate pupae (about 3 h before release) and pupae that had ecdysed less than 1 h before sacrifice (i.e. less than h after hormone release). The relative amounts of eclosion hormone activity in these two regions of the CNS is illustrated in Fig. 1. The brains from AS stage pharate pupae and from pup showed similar amounts of extractable activity. By contrast, the activity in the abdominal nerve cord dropped by approximately 50−75 % during this h period. Thus, the hormonal activity that appears in the blood before ecdysis apparently comes primarily from stores in the abdominal ganglia. The thoracic ganglia probably also contribute to this release but this was not tested.

In order to confirm that the prepupal brain was not the source of the blood-borne eclosion hormone activity, we removed brains from larvae that had just entered the wandering stage (about 4 days before the scheduled time of pupal ecdysis). These animals were somewhat delayed in their subsequent timetable of pupal development because the brain extirpation also removed the source of the prothoracicotropic hormone that normally drives ecdysone secretion (Williams, 1952). As seen in Table 2, over 60% of the operated animals went on to show a complete pupal ecdysis behaviour and shed the old larval cuticle. These animals that underwent successful ecdysis did so at the proper stage of pupal development as measured by the state of tanning of the pupal cuticle (see timetable in Truman et al. 1980). Of the remaining debrained animals, a number showed at least the initiation of the ecdysis programme as seen by the pulling out of the tracheal linings in the posterior abdominal segments. Interestingly, even the animals that did not show a complete ecdysis nevertheless eventually turned ventral-side-up thereby assuming the characteristic pupal posture.

To verify the assumption that debrained animals performed the behaviour in response to blood-borne hormone, we bled five such animals just as they began ecdysis movements. The individual blood samples from each of these animals scored strongly positive in the pupal ecdysis assay; the mean score was 143 ± 10 (± s.E.). This compares favourably with the mean score of 171 obtained from blood of ecdysing intact animals (Truman et al. 1980). Therefore although the pharate pupal brain contains substantial amounts of eclosion hormone activity, it appears to have little or no function in the secretion of the hormone that triggers pupal ecdysis.

The ecdysis stimulating activity in the pharate pupal brain and abdominal nerve cord was characterized as to its apparent molecular weight and charge. Heat-treated extracts of brains and abdominal nerve cords were subjected to gel filtration chromatography through a Sephadex G-50 column. The fractions were then assayed for eclosion hormone activity using the pharate adult Manduca isolated wing assay iReynolds, 1977). As seen in Fig. 2, both tissues showed a large peak of activity that centred around a V_e/V_o ratio of about 1·6, which corresponded to an apparent molecular weight of 8000−9000 daltons. Assay of these active fractions on H. cecropià isolated abdomens showed that those with substantial wing plasticising activity also were capable of releasing various behaviours of the adult emergence sequence. Fig. 2 also shows that the extracts from both tissues showed only one peak of pupal ecdysis stimulating activity and that in both cases it coincided with the eclosion hormone activities revealed by the other two assays.

The active fractions from the Sephadex G-50 column were then subjected to wide band (pH 3−9) isoelectrofocusing. In both the brain (Fig. 3 A) and the abdominal nerve cord (Fig. 3 B) the various biological assays revealed only one peak of activity at approximately pH = 5. Thus, the eclosion hormone activities present in the brain and abdominal CNS of pharate pupae have common molecular properties in so far as they have similar molecular weights and isoelectric points.

In pharate adult moths eclosion is triggered by the release of eclosion hormone that had been made in brain neurosecretory cells during adult development and transported down the axons to terminals in the CC for subsequent release (Truman, 1973). An initial survey of larvae and pupae for eclosion hormone activity (Truman, 1973) concluded that the brain-CC-CA complexes from these stages contained at most 10% of the amount of hormone found in the pharate adult. This estimate was made using a whole-animal behavioural assay which could only detect amounts of hormone as small as this with some degree of uncertainty. The conclusion drawn at the time from this finding was that eclosion hormone was unlikely to be important in the control of ecdysis in larvae and pupae.

The results of the present study require that this conclusion be reevaluated. The initial estimate of eclosion hormone content in the pharate pupal brain has been confirmed here (0·07 units of eclosion hormone). In addition we also find 0·14 units stored in the CNS outside of the brain, in the thoracic and abdominal ganglia. The total amount of eclosion hormone in the pharate pupa is thus about 21 % of that in the pharate adult CC, but this smaller quantity of hormone assumes increased significance with the finding that the pharate pupal CNS is approximately 10 times more sensitive to the eclosion hormone than is that of the pharate adult (Truman et al. 1980).

The distribution of activity in the pharate pupal CNS posed two questions: are the activities in the brain and the ventral nerve cord attributable to the same factor, and is either, or both, the same as the eclosion hormone of the pharate adult? The results of gel filtration chromatography and electrofocusing showed that both tissues contained only one biologically active species and that the apparent molecular weight and charge of the active factor were the same in each case. Both factors were active in all three bioassays. The total amount of activity in the active fractions after gel filtration chromatography (Fig. 2) was calculated using the standard dose response curves for each assay (Reynolds & Truman, 1980; Truman et al. 1980; Mumby, Truman & Reynolds, in preparation). A value was then obtained for the number of 50% dosages that were present in each extract for each assay. The ratio of 50% dosages for adult abdomen eclosion: pupal ecdysis : adult wing plasticization was 45 : 1 :27 for prepupal brains and 0·37:1:30 for the abdominal nerve cord. Thus, e activities from the 2 parts of the pharate pupal CNS were very similar in two aspects of their molecular properties and in their relative potencies in three different biological assays. We conclude that it is highly likely that these activities are due to the same molecule.

Eclosion hormone purified from heads of pharate adult Manduca (Reynolds & Truman, 1980) or from CC-CA complexes from the same stage (Mumby, Truman & Reynolds, in preparation) has an apparent molecular weight of 8500 daltons. Wide band electrofocusing (pH 3·9) shows one band of activity centring around 4·9 (Reynolds & Truman, 1980). When the ratios of 50% dosages for pharate adult CC-CA extracts, or for electrofocused eclosion hormone from the same source are computed for the three assays listed above, ratios of 0·12:1:13 and 0·06:1:16 respectively are obtained (Reynolds & Truman, 1980; Truman et al. 1980; Mumby, Truman & Reynolds, in preparation). Thus, purified adult eclosion hormone preparations have a spectrum of biological activity that is similar to the material obtained from pharate pupae. Also, the molecular properties of hormone from the two stages are very similar. Consequently we conclude that the prepupal activity is probably identical to the eclosion hormone of the pharate adult and that the same hormone is used to trigger both pupal ecdysis and adult eclosion. Definitive confirmation of this conclusion will require amino acid sequence determinations of hormone purified from each of the two stages.

We have presented two lines of evidence to suggest that, in the pharate pupa, eclosion hormone is released from the ventral nerve cord rather than from the brain, as is the case in the pharate adult. First, the store of hormone in the pupal abdominal CNS is depleted during the course of ecdysis, whereas that in the brain is not. Second, debrained insects release eclosion hormone into their blood in approximately normal quantities and undergo pupal ecdysis at the proper developmental stage. It is significant that although the pharate pupal brain contains appreciable amounts of eclosion hormone, we could find no activity in the brain’s neuro-haemal organ, the corpora cardica. In the pharate adult on the day of eclosion, the CC contains the majority of the total cephalic store of eclosion hormone (Truman, 1973).

Why does the pharate pupal brain contain a store of eclosion hormone which is not destined for release at pupal ecdysis? The eclosion hormone present at this time may simply represent an early stage in the process whereby hormone is accumulated for future use at adult ecdysis. Alternatively, the store of hormone in the brain may not be destined for release into the blood but may represent a local, non-hormonal, peptidergic pathway within the brain. A number of studies have shown that hormones which are secreted into the blood may also be released locally in the CNS to modulate neuronal activity. Examples include the egg-laying hormone of the mollusc Aplysia (Branton et al. 1978) and the luteinizing hormone releasing hormone of amphibians (Jan, Jan & Kuffler, 1979).

A distribution of hormonal activity between the brain-CC-CA complex and the chain of ventral ganglia has been reported for a number of different insect hormones íraenkel & Hsiao, 1965; Gersch & Sturzebecher, 1967; Hartmann, Wolf & Loher, 1972). In the case of the last example, a hormone that promotes oocyte maturation in a grasshopper, Gomphocerus rufus, it was suggested that the ventral ganglio centre supplemented the brain centre and could partially compensate for damage to the pars intercerebralis (Hartmann et al. 1972). The present study shows that eclosion hormone has a broad distribution in the CNS but that the two centres are controlled independently and are used at different times in the life history of the insect.

The existence of two distinct sites for eclosion hormone release presents the question of what are the factors responsible for selection of the brain or the ventral nerve cord sites. Adult eclosion in silkmoths and in the sphinx moth, Manduca, is controlled by a circadian clock that is sensitive to light (Truman, 1972 a) or temperature cycles (Lockshin, Rosett & Srokose, 1975) respectively. In the silkmoths the circadian system comprised of a photoreceptor and clock is contained within the brain (Truman, 1972 a) and directs the release of eclosion hormone from its brain centre. Other neurohormones in insects that are released under photoperiodic control are likewise released from endocrine centres in the brain (Williams & Adkisson, 1964; Steele & Lees, 1977; Truman, 1976). In contrast to the above examples, pupal ecdysis in Manduca is similar to larval ecdysis (Truman, 1972b) in that the event itself is not gated but rather is developmentally triggered. In this respect it is of interest that for this ecdysis, the hormone is released from the ventral nerve cord rather than from the brain. From this limited set, one would predict that the brain centre presides over photoperiodically gated releases of hormone whereas the ventral nerve cord centre is reserved for those triggered by endogenous, developmental factors. It will be of interest to determine if this association between the site of hormone release and a gated or developmental control over release will hold true for ecdyses of other stages and of other insects.

We thank Mr Oliver Dominick for advice on the surgical techniques. These studies were supported by grants from NSF (PCM 77−24878) and from NIH (Roí NS 13079 and K04 NS 00193). P.H.T. was supported by a NIH training grant (GM 0710805) and by an NSF Predoctoral Fellowship (63−6665).