J. Zool., Lmd. (1981) 194, 143-163

Photoperiod and larval body size: integrated factors controlling onset of the moulting cycle in Heliconius rnelpornene (Lepidoptera)

SHEILA WRIGHT A N D KENNETH U. CLARKE University o j Nottingham, University Park, Nottingham

(Accepted 10 June 1980)

(With 7 figures in the text)

In Heliconiirs melpomene, ecdysis to each stadium is asynchronous in continuous light, but is synchronized with respect to the time of day in an LD 12 : 12 photoperiod regime-generally occurring during the early part of the photophase. This is a consequence of synchronism in the time of day of onset of the moulting cycle, which occurs approximately 24 hours before ecdysis. There is variation in the number of days taken to complete an instar, however. Onset of the moulting cycle is gated by photoperiod, and occurs at the first gate following attainment by the larvae of a certain threshold size, indicated by weight. By depriving larvae of food, the time at which threshold size is obtained can be delayed; this results in a delay in the time of onset of the moulting cycle. Possible biological advantages in the wild of the existence of a threshold size for onset of the moulting cycle, and of synchronism in the time of day of ecdysis, have been suggested.

Contents Page

Introduction. . . . . . . . . . . . . . . . . . . . . . 143 Materials and methods . . . . . . . . . . . . . . . . . . 143 Results . . . . . . . . . . . . . . . . . . . . . . 145 Discussion . . . . . . . . . . . . . . . . . . . . . . 154

References . . . . . . . . . . . . . . . . . . . . . . 161 Summary . . . . . . . . . . . . . . . . . . . . . . 160

Introduction Much work has been done on the systematics, genetics, and external morphology of the

Heliconiini but surprisingly there have been few studies of the physiology of this interesting group of Lepidoptera-and no work has been published on their hormonal system. In an attempt to fill this gap in our knowledge of the heliconids, an investigation of some of the factors controlling the moulting cycle of Heliconius melpomene was conducted.

Materials and methods Heliconius melpomene race thelxiope was used as the experimental animal. It is native of the

rainforests surrounding the Brazilian city of Belem at the mouth of the Amazon. Given suitable conditions, it can be reared with ease in captivity (Turner, 1974).

The stock culture was housed in a large rearing cage, inside a humid greenhouse heated to 28°C. The butterflies were fed with a dilute honey solution (recommended by Turner, 1974). Since butterflies of the genus Heliconius are unique in having a requirement for pollen in the diet (Gilbert, 1972), flowering plants with a high pollen content were also provided-those in the family Curcurbitaceae are particularly favoured by these butterflies. The larvae of the Heliconiini

143 0022-546018 I /060143 +21 $02.00/0 6 198 I The Zoological Society of London

144 S . WRIGHT AND K . U. CLARKE

feed upon various members of the Passifloracae-the co-evolution that has occurred between these butterflies and Passion vines has been the subject of an investigation by Benson, Brown & Gilbert (1976). Passijlora caerulea proved to be a suitable food plant for H. melpomene, and females oviposited readily upon the fresh shoots and tendrils of growing plants which were placed around the rearing cage. Heliconiusfemales are long-lived andlay afew eggs each day throughout their lives.

Since environmental conditions in the stock culture were unavoidably somewhat variable, larvae required for experimental purposes were reared from the egg in an insectary where the environ- ment could be more rigorously controlled. The temperature was maintained at 28 f- 1”C, and the relative humidity at 70 k 5 %. Illumination was by fluorescent strip lighting over the rearing cages, linked to a Venner time switch to give a photoperiod of 12 h light, 12 h dark (LD 12 : 12), with “lights on” at 0830 hrs and “lights off” at 2030 hrs.

301 Instar II-m I

30 Instar III-IV

20

‘Z 301 lnstor W-V

20

30 Instar V- pupa

2 4

l o h T r m - m d 0030 0430 0030 1230 1630 0230

Time of day (hrs)

FIG. 1 . The time of day of ecdysis to each stadium in continuous light.

For experiments which necessitated manipulation of the photoperiod regime-a photoperiod control cabinet was employed. This consisted of three light-proof compartments in each of which the photoperiod could be independently controlled. Each compartment was illuminated by a centrally placed vertical fluorescent tube. Temperature, humidity and light intensity with the cabinet were as for the insectary. All experimental larvae were fed on growing PassiJora caerulea. Under the conditions outlined, development was rapid, taking only 3-4 weeks from egg to adult butterfly. Of this 24-38 days were spent as an egg, 12-17 days as a larva (there are five larval instars), and 8 days in the pupal stage.

Some of the experimental work involved ligaturing larvae. This was done by passing a cotton thread around the body, either between the head and prothorax or between the metathorax and the first abdominal segment. A double knot was tied in the cotton to secure it. A similar technique has been used successfully by many workers on insect hormones (for example on dipterous larvae by Price, 1970, and Zdarek & Fraenkel, 1971; and on lepidopterous larvae by Fukuda, 1944;

CONTROL O F MOULTING I N H . MELPOMENE 145

Kobayashi, 1952, and Weir, 1970). After ligation, animals were placed in isolation until the follow- ing day-when they were examined to determine the effects of the procedure. If new cuticle had formed in any section of the body, the unshed exuvia could be peeled away with forceps to reveal the new cuticle beneath. This procedure was usually facilitated by the copious supply of moulting fluid between the old and new cuticles.

Results When larvae of Heliconius melpomene were reared from the egg in either continuous

light or continuous darkness, ecdysis occurred asynchronously throughout each 24 h period, as shown in Fig. 1. When the larvae were reared under conditions of 12 h light followed by 12 h darkness, however, an entirely different pattern emerged. In a photoperiod regime with “lights on” at 0830 hrs and “lights off” at 2030 hrs, the time of day at which larvae cast their cuticles was synchronized. Of the larvae undergoing ecdysis on a particular day, most did so within a few hours of each other-in the early part of the photophase close to “lights on”, with some larvae moulting a little before but most a little after this event. This was true of ecdysis to each of the stadia monitored, as shown in Fig. 2, although that to instar V tended to occur a little later on average than those to the other stadia.

Scotophose Photophose

11nstar111-IV

$ 3ollnstor N-v I e I

lnstar V- pupa

40 :tll , I i i i liii 2 0

10

0030 0430 0830 1230 T-R 1630

7 2030

Time of day (hrs)

FIG. 2. The time of day of ecdysis to each stadium in an LD 12 : 12 photoperiod regime, with “lights on” at 0830 hrs.

I46 S. W R I G H T A N D K. U . C L A R K E

If larvae were reared in an LD 12 : 12 photoperiod regime in which “lights on” was shifted by eight hours, ecdysis became synchronized with respect to this regime in the same way as to the regime in which “lights on” occurred at 0830 hrs-with the mean ,time of ecdysis to each instar being shifted by approximately eight hours.

Larvae maintained in an LD 18 : 6 regime moulted at a similar time with respect to “lights on” as those reared under a photoperiod of LD 12 : 12. This indicated that it is probably the time at which “lights on” occurs that determines the time of day at which ecdysis will occur, rather than the total length of either the photophase or the scotophase.

Although in an LD 12 : 12 photoperiod regime, larvae underwent ecdysis to each stadium at a similar time of day to one another, there was variation in the number of days taken to complete an instar. Those larvae failing to undergo ecdysis in the late scotophase or early photophase of a particular day did not do so until this time on the following day, or even on the one after that-thus the time at which ecdysis occurred was gated. Normally, either two or three days were spent in instar 111; two, three or four in instar IV, and four, five or six in instar V, although occasionally larvae which spent a longer period in an instar were encountered. Table 1 shows the proportion of larvae taking each number of days to complete instars, III ,IV and V out of a group of 103 reared from the egg under a photoperiod regime of LD 12 : 12. Tt can be seen that in instar 111 most larvae spent two days (87.5%) in instar IV, three days (68.1 %) and in instar V, four days (62.5%). The majority of animals thus spent a total of nine days in instars 111 to V inclusive, although the total duration of these three instars as well as their individual length was variable- and ranged from eight to 12 days.

T A B L E I

Duration of instar (days) Instar 2 3 4 5 6 7

N o significant difference was found between the numbers of male and female larvae of any particular instar length-the sex of a larva appeared to have little influence either upon the number of days spent in an instar or upon the total duration of larval life.

As ecdysis to any stadium generally occurred at a similar time of day as that to the previous one, the time spent by a larva in each closely approximated to a complete number of days-as shown in Fig. 3. For each stadium, larvae will henceforth be termed two, three or four-day animals etc., according to the length of time spent in that stadium. Two, three and four-day IVth instar larvae all underwent ecdysis to instar V at a rather similar time of day, as indicated in Fig. 4, although two-day animals moulted on average a few hours later in the day than either three or four-day larvae. At this point, two problems required investigation. Firstly, at what stage were the moulting cycles of individual larvae being brought into synchronism with respect to the time of day? Secondly, why did larvae vary in the number of days taken to complete an instar?

Most of the work that follows was performed on IVth instar larvae-since these show

CONTROL O F M O U L T I N G I N H . M E L P O M E N E 147

--I 20 I Instar IU

!ii-J 5

2 4 48 72 96 I20 144

Duration of lnslar (h)

FIG. 3. The number of hours spent by larvae in instars 111, IV, and V.

30

I3-day larvae

I 2 0

i 4-day larvae

2 0

TT3 0830 1230 1630 C 30

Time of day (hrs)

FIG. 4. The time of day of ecdysis to instar V of two, three, and four-day IVth instar larvae.

148 S . WRIGHT A N D K . U. C L A R K E

the most pronounced polymorphism in instar duration. In addition, by using IVth instar larvae in preference to Vth, complexities in the moulting cycle of the latter associated with the larval to pupal transformation were avoided.

It is conceivable that animals may at any time of day become competent to undergo ecdysis, with only this final stage of the moulting cycle, i.e. the splitting and casting of the old cuticle, being restricted to a particular period. On investigation of events in instar IV, however, it became apparent that this was not the case. Usually, the first externally visible sign that the moulting cycle of a lepidopterous larva is underway occurs at a point approxi- mately midway through the process of new cuticle formation, when feeding ceases and the old head capsule slips forward to reveal the partially formed new one beneath the cuticle of the first thoracic segment-over which it comes to rest as a “muzzle”. After this has occurred, the larva remains motionless until ecdysis. It was observed that in Heliconius melpomene reared in an LD 12 : 12 photoperiod regime, head capsule slippage generally occurred during the last few hours of the photophase. This observation was quantified as follows. A number of larvae were randomly selected at the beginning of instar IV, and both the time of day at which the head capsule slipped, and the day of the instar upon which this occurred, were recorded for each animal. Finally the time at which each larva underwent ecdysis to instar V was recorded as accurately as possible. The results are summarized in Table 11. The time at which head capsule slippage occurred ranged from 1600 to 2100 hrs on the evening before ecdysis, showing that this event was indeed syn- chronized with respect to the time of day, as was ecdysis. In three-day larvae, head capsule slippage occurred a whole day later than in two-day ones, the animals continuing to feed and grow for approximately a further 24 h. Similarly, in four-day larvae, head capsule slippage and cessation of feeding occurred a day later than in three-day larvae. Thus, either head capsule slippage itself, or some stage in the moulting cycle prior to this event, was being synchronized by photoperiod, and not merely the time of ecdysis. Furthermore, the length of time elapsing between the time an individual slipped its head capsule and the time that it underwent ecdysis varied within only a narrow range-from 16 to 18 hours (mean 16.8 h). Animals which slipped the head capsule at a later time of day underwent ecdysis to instar V at a correspondingly later time on the following morning, indicating that there is unlikely to be a separate mechanism to control the time of ecdysis in addition to that which synchronizes some earlier event in the moulting cycle. Rather, ecdysis follows at a fixed number of hours from this event-the duration of this period being determined only by the length of the physical and chemical processes involved in the completion of new cuticle formation.

TABLE I1

Time of head capsule Duration slippage of instar IV Mean k S.D. Range

(days) (hrs f h. min) (hrs)

~ ~____

Time of day of ecdysis to instar V

Mean & S.D. Range (hrsf h. min) (hrs)

Time elapsing between head capsule slippage and ecdysis Mean f S.D. Range

(h) (h ) ~~ ~ ~

2 19 I0 k 0.53 1800-2000

4 1915f 1.12 1730-2100 Total 1821 f 1.41 1600-2100

3 1730k 1.36 1600-2100 1159k0.44 1120-1245 101 1 k2.05 0837-1405 1217k 1.29 1105-1435 1109+2.04 0837-1435

16.83k0.29 16.17-17.49 16.69&0.41 16.00-17.75 17.04f0.67 16.08-18.00 16.80 f 0.47 16.00-1 8.00

CONTROL OF MOULTING IN H . M E L P O M E N E 149

The next step in the investigation was to discover whether the moulting cycle of H. melpomene was brought into synchronism with respect to the time of day at head capsule slippage, or at an earlier stage-perhaps through the influence of photoperiod upon one or more of the hormones responsible for initiating the proces of new cuticle formation. Ligaturing experiments on H . melpomene larvae were carried out to investigate the timing of hormonal events in the moulting cycle of two- three- and four-day IVth instar animals. First, however, it was necessary to find a method of distinguishing between two- three- and four-day IVth instar larvae at a stage preceding head capsule slippage. Preliminary observa- tions indicated that the higher the rate of weight increase of the larvae, the shorter the number of days spent in the instar. To quantify this, a number of larvae were weighed at regular intervals throughout the IVth instar, and the rate of‘ weight increase calculated for each. The mean regression lines for the rate of weight increase in two-, three- and four-day larvae are shown in Fig. 5 . By calculating the rate of weight increase of an individual it was therefore possible to predict with some accuracy how many days the larvae was likely to take to complete instar IV. Absolute weight of larvae at certain times during the instar was also found to give a good indication of whether the larvae would be two-, three- or four- day animals. At 24 h, for example, the majority of two-day larvae weighed considerably more than three-day animals, and at 48 h three-day larvae were heavier than four-day ones.

In Heliconius melpomene reared in a photoperiod regime of LD 12 : 12 with “lights on” at 0830 hrs, head capsule slippage occurs between 1600 hrs and 2100 hrs, some 16-18 h before ecdysis. In order to determine when the critical period for ecdysone occurs, and

160

140

- ?

- 3-day

150 S . WRIGHT AND K. U. C L A R K E

whether this too is synchronized with respect to the time of day, groups of larvae were ligatured between the thorax and abdomen at regular intervals during the hours preceding head capsule slippage. Initially, four groups of eight IVth instar larvae (selected from those animals which had the high rate of weight increase characteristic of two-day larvae and were heavy in weight at 24 h) were ligatured at two hourly intervals between 1000 and 1600 hrs on the day on which head capsule slippage was due to occur in two-day larvae. The experiment was repeated on the following day of the instar on four groups of eight three-day larvae. The results are shown in Table 111. In two-day larvae, the critical period for ecdysone (Table 111) ends between 1400 and 1600 hrs on the day preceding ecdysis, since at 1400 hrs a ligature placed between the thorax and abdomen prevented new cuticle formation in that part of the body posterior to the ligature, whereas at 1600 hrs it did not. Likewise for three-day larvae, the end of the critical period for ecdysone is synchronized with respect to the time of day and in the majority of animals (87.5 o{, ends between 1000 and 1200 hrs on the day preceding ecdysis, i.e. some 20 h later than in two-day larvae. Since the end of the critical period for ecdysone is synchronized, and since ecdysone action upon the epidermis is a chemical process, which presumably takes a fixed time to complete under constant environmental conditions, it seems reasonable to assume that the release of ecdysone is also synchronized with respect to the time of day. It can be seen from Table 111 that in all but one of the two-day IVth instar larvae (87.5%), ligation between the thorax and abdomen at 1000 hrs completely prevented new cuticle formation, both anterior and posterior to the ligature. This indicates that the abdomen may in some way be involved in moulting cycle initiation. Possibly, some stimulus from the abdomen is required to initiate prothoracotropic hormone (PTTH) release from the brain-corpus cardiaca system, although further work is necessary to show whether or not this is the case. Nevertheless, the fact that ligation of two-day larvae at 1000 hrs can block new cuticle formation indicates that PTTH release has probably not occurred by this time in the majority of animals. In view of this result a group of eight three-day larvae were ligatured between the thorax and abdomen at 0800 hrs on the day before ecdysis was due to occur. In 62.5 % of these animals too, new cuticle formation was prevented completely. (In the remaining 37.5 %, it occurred anterior to the ligature only.) Thus, in the majority of three-day larvae, PTTH release occurs between 0800 and 1000 hrs. To determine whether the critical period for PTTH ended between 0800 and 1000 hrs or between 1000 and 1200 hrs in three-day larvae, a further group of eight were ligatured between the head and prothorax at 1000 hrs. In all but one of the larvae, new

T A B L E 111 ~ _ _ ~ _ _ ~ ~

Time of day 2 day IVth instar larvae of ligation (-*----,

(hrs) 00 +o + + 3 day IVth instar larvae

00 + o + + 7

1000 87.5% 12.5% 07, 1200 2576 75% 07, 1400 0% 100% 0 ; ; 1600 0% 0% loop,

~~

00 = new cuticle formed neither anterior nor posterior to the ligature. O+ = new cuticle formed anterior to the ligature only. + + = new cuticle formed both anterior and posterior to the ligature.

CONTROL O F M O U L T I F G I N H . M E L P O M E i Y E 151

cuticle formed in the region of the body posterior to the neck ligature. This shows that in the majority of three-day animals, the critical period for PTTH has ended by 1000 hrs- less than 2 h after its release. It is interesting to note that in none of the larvae neck- ligatured at 1000 hrs was the new cuticle of the pupal type. This contrasts with the results obtained by Fukuda (1944) on Bombyx mori, and by Nijhout & Williams (1974u, 6) on Munducu sextu, each of whom were able to detect a critical period for juvenile hormone (JH). Neck ligation before the end of this period resulted in formation of a pupal-type cuticle posterior to the ligature, rather than a larval one, at what would normally have been a larval to larval moult. The results obtained for H. melpomene suggest that the critical period for JH is very short, ending between 0800 and 1000 hrs in three-day IVth instar larvae, as does the critical period for PTTH.

PTTH release and the end of the critical period for ecdysone both occur several hours later with respect to the time of day in two-day IVth instars than in three-day animals. This correlates with the observation made earlier that both head capsule slippage and ecdysis occur on average a few hours later in the day in two-day larvae (the mean time of ecdysis to instar V is 14-02 for two-day larvae and 11.17 for three-day ones). It is additional evidence that the time of ecdysis is determined entirely by the timing of early hormonal events in the moulting cycle.

Ligature of four-day IVth instar larvae revealed that hormonal events in the moulting cycle occur at a similar time of day to those in three-day larvae, but a day later.

To summarize, it is synchronization with respect to the time of day of the action of PTTH on the prothoracic glands, and even of PTTH release from the brain-corpus cardiaca complex, that results in synchronization of ecdysis some 24 h later.

To return now to the question of why larvae vary in the number of days spent in an instar. As stated earlier it is possible to distinguish between two-, three- and four-day lVth instar larvae both by their rate of weight increase and by their absolute weight at certain times during the IVth instar. This implies that the size of a larva may play an important part in moulting cycle initiation and therefore in determining how many days it will spend in the IVth instar. Alternatively, two-, three- and four-day larvae may represent three distinct morphs, each genetically programmed to spend a fixed number of days in the instar, and each having a different potential rate of weight increase associated with it. To test these possibilities, a number of larvae were deprived of food for various periods of time during instar IV, to reduce both their rate of weight increase and their absolute weight at given times during the instar, in comparison with larvae that were allowed to feed normally. If either rate of weight increase or absolute size are important in determining when moult- ing cycle initiation takes place, then food deprivation should prevent or delay onset of the moulting cycle. If, however, larvae are genetically programmed to spend either two-, three- or four-days in instar IV-then food deprivation should result in the larvae undergoing ecdysis to instar V at the time at which they normally would, but at a reduced size. The results of this experiment showed that moulting cycle initiation was delayed until larva had exceeded a threshold size (indicated by a weight of 65-75 mg) by the time of day at which PTTH release can occur. Of 18 larvae that were alternately fed and deprived of food during instar IV, 16 had exceeded 75 mg in weight 24 h before their eventual ecdysis to instar V, yet were below 65mg 48 h before ecdysis. (As shown earlier-PTTH release occurs approximately 24 h before ecdysis.) Several of these animals remained in the IVth instar for as long as nine days before attaining threshold size and undergoing the moulting

I52 S . W R I G H T A N D K . U . C L A R K E

cycle that culminated in ecdysis to instar V. A further group, consisting of eight larvae, were, by food deprivation, prevented from reaching 65 rng in weight. All eventually died without showing any sign of new cuticle formation.

The results indicate that it is absolute size rather than rate of weight increase that determines when moulting cycle initiation will occur. Rate of weight increase is very low under conditions of alternate feeding and food deprivation, yet larvae still undergo moult- ing cycle initiation at the first gate following attainment of threshold size. Figure 6 shows the weights of a group of normally fed IVth instar larvae at 1000 on the second day of the instar-when they were 24 f 1 h old, i.e. at approximately the time at which PTTH release occurs in two-day larvae. In these animals too, there appeared to be a threshold size indicated by a weight of 65-75 mg above which moulting cycle initiation was triggered (two- day larvae), and below which the larvae had to wait at least until the following day before moulting cycle initiation could occur (three- and four-day larvae). Figure 7 shows the weight of the larvae at 1000 on the third day of the instar-when they were 48 I 1 h old. Once again, those larvae over the threshold size were triggered to undergo moulting cycle initiation at this time (three-day larvae), the remainder went on feeding and growing for a further 24 h (four-day larvae). At 72 h, all of these larvae exceeded 80 mg in weight, and underwent ecdysis on the following morning. From a comparison of Figs 6 and 7, it appears that the threshold weight is somewhat higher at 48 h than at 24 h-in the region of 70-80 rng. This is probably an apparent rather than a real difference-caused by the fact that although on both days larvae were weighed at 1000 hrs, moulting cycle initiation in two-day larvae occurs on average a few hours later than in three-day larvae. Thus, the majority of two-day larvae are being weighed a little before moulting cycle initiation and

35

30

2 5

2 0 -

15 -

10- - 5 - e 0 L -

4 0 t 2-day lorvae - - -

I ; c 3-and4-doy larvae

30

25 50 75 100 125

Weight (rng)

FIG. 6. The weight of lVth instar larvae at 24 h.

CONTROL OF M O U L T I N G I N H . M E I - P O M E N E 153

three-day larvae a little after this event, accounting for the apparently higher value for threshold weight in the latter. The reason why moulting cyc:le initiation in two-day larvae occurs on average a few hours later in the day than in three-day animals may simply be that the gate at which they are triggered opens at a later time of day than that at which three- day larvae are triggered. However, some evidence for an alternative possibility was obtained. It was found that the heavier a two-day larva is at 1000 hrs on the second day of the instar, the earlier the time of ecdysis on the following day. Those larvae which were only just over 60 mg at this time formed the latter end of the range of several hours that occurred in the time of day at which two-day larvae underwent ecdysis. This can be explained in terms of a gate which extends for several hours. It may open at the same time on the second day of instar IV as on the third-but on the second day many of the two-day larvae have not attained threshold size by the time that the gate opens, and instead do so during it. The three-day larvae, which do not attain threshold size by the time that this first gate closes, even if they do so shortly afterwards must wait for a further day before moulting cycle initiation can occur. During this time they continue to feed and grow, and thus when the gate re-opens many are well above the threshold size and may be a good deal heavier than most of the two-day larvae were at the previous gate. In the three-day larvae, moulting cycle initiation thus occurs soon after the gate re-opens, and they undergo ecdysis at a correspondingly earlier time on the following day. Table IV shows that the mean weight of two day larvae at 1000 hrs on the second day of the 4th instar is only 66.52 mg, whereas that of three-day larvae at 1000 hrs on the third day is 98.97 mg. This difference in weight, incidentally, is to some extent reflected in a higher mean weight of three-day larvae at ecdysis to instar V, in comparison with two-day animals. However, the latter have on average a higher rate of weight increase than the former, and thus-during the period of feeding which occurs between moulting cycle initiation and head capsule slippage, to some extent compensate for the difference in weight seen between the two at the onset of the

30

30 - 25 - 2 0

15-

10-

5-

-

I I I I I I I 11-

25 50 75 100 125 15C

Weight (mg)

FIG. 7 . The weight of lVth instar larvae at 48 h.

154 S . W R I G H T A N D K . U . C L A R K E

moulting cycle. The four-day larvae reach about the same mean weight at ecdysis as the three-day larvae.

Although most of the experimental work was restricted to IVth instar larvae, it seems likely that a similar gating mechanism to that discovered in instar IV exists in other instars too-whereby larvae must reach a threshold size before moulting cycle initiation can occur. As in instar IV, two-day IIIrd instar larvae are heavier at 24 h than three-day ones, and there appears to be a threshold weight of approximately 15 mg. In the Vth instar, the time at which onset of the moulting cycle occurs is unclear. In the final instar of a number of lepidopterous larvae, not one but two periods of a-ecdysone release occur, e.g. in Manduca sexfa (Riddiford, 1976), Pievis brassicae (Lafont, Mauchamp, et a/., 1975) and Bombyx iiiori (Shaaya & Karlson, 1965). However, the weight of the majority of four-day Vth instar larvae at 48 h is above that of five-day animals, and the weight of five-day larvae at 72 h is above that of six-day ones, indicating that a similar mechanism to that found in instar IV is probably in operation.

Little relationship seems to exist between the number of days spent in one instar and the number spent in the next. Out of a group of lVth instar larvae, the numbers of two-, three- and four-day animals spending four, five or six days in instar V were recorded and are given in Table V. It can be seen that both two- and three-day lVth instar larvae gave rise to similar proportions of four- and five-day Vth instar larvae. (The numbers of four-day lVth and six-day Vth iiistar animals in the group were not large enough for any relation- ships to be drawn.)

T A B L E 1 V

Duration of Number of Mean weight (mg)? s.d. instar IV larvae Day 2 Day 3 Day 4 (days) weighed (at 1000 hrs) (at 1000 hrs) (at 1000 hrs) Pre-ecdysis

2 17 66.52 f 11.02 ~ ~ 87.05 13.22 3 60 50.68 f 10.55 98.97 +_ 18.20 - 98.161 14.14 4 11 40.71 k4.77 67.39 f 1.71 101.02f7~51 94.12 & 6.95

T A B L E V

Duration of instar IV No. of Duration of instar V (days)

(days) larvae 4 5 6

Discussion To summarize, in each instar, the moulting cycle of H. melpomene cannot proceed until

the larva has attained a threshold size. Onset of the moulting cycle is gated by photoperiod. Even in larvae which have exceeded the threshold size, initiation of new cuticle formation normally only occurs during the early part of the photophase. Ecdysis occurs approxi- mately 24 h after onset of the moulting cycle, as a consequence of synchronization of onset of the moulting cycle, it too is synchronized with respect to the time of day.

C O N T R O L O F MOULTING I N H . MEl .PO.MENE 155

It is apparent that mechanisms must exist in the larvae both for detecting their body size and signalling that threshold size has been attained, and for perceiving the appropriate cue from the photoperiod regime which gates PTTH release. The information from each of these sources must then be integrated before PTTH release can occur.

How is body size detected by the larvae of H. melpomene and how is the attainment of threshold size signalled? In Rhodnius prolixus, stretching and distension of the abdominal cuticle during a blood meal stimulates stretch receptors which are responsible for triggering PTTH production and release (Van der Kloot, 1961). Is it possible that in H. melpomene a similar mechanism may signal attainment of threshold size ? Although no sudden distension of the cuticle over a period of minutes occurs in this species, there is a great deal of cuticular distension during the course of an instar, which may be sufficient to trigger the production of PTTH when reaching a certain threshold value. In instar IV, for example, body weight may increase five- or even six-fold, from around 20 mg to 100-120 mg or even more in some three-day larvae. A four- or five-fold increase in weight occurs between ecdysis to instar IV and attainment of threshold size at 65-75 mg. Hepburn & Levy (1975) and Hepburn & Chandler (1976) showed that in the silkworm Rombyx mori, not only does a filling out of folds in the cuticle occur during the course of’ an instar, i.e. distension, but there is in addition, actual stretching of the cuticle, involvirig mechanical hysterisis of the protein molecules of which it is composed. In experiments in which the cuticle of B. mori was stretched artificially, these authors showed that work-hardening of the cuticle occurred, i.e. the more the cuticle was stretched, the more resistant it became to further stretching. Hepburn & Chandler (1976) tentatively suggested that this work-hardening effect may be the basis of the mechanism in some species by which proprioreceptors are recruited to signal when onset of the moulting cycle should occur. There is as yet no direct evidence that cuticular distension or stretch is important in initiating the moulting cycle of any insect species other than Rhodnius prolixus, however. Such conclusions must therefore be specula- tive at this stage, although the discovery that in H. melpomene and in Manduca sexta (Truman, 1972) a threshold size exists for onset of the moulting cycle lends support to the suggestion forwarded by Hepburn & Chandler.

In having to attain a threshold size before moulting cycle initiation can occur, larvae of H. melpomene differ greatly from those of the clothes moth Tineola biselliella (Titschack, 1926), the wax moth Galleria mellonella (Metalnikov, 1908; Allegret, 1964) the corn borer Ostrinia nubilalis (Beck, 1950) and the meal worm Tenebrio molitor (cited by Wigglesworth, 1970), all of which if deprived of food will undergo a series of static ecdyses and may eventually metamorphose to adult insects of a very small size. Adults of Galleria mellonella of only a tenth of the weight of normal individuals have been produced by starving the larvae for periods during their development. In contrast to the species cited above, larvae of the tobacco hornworm, Manduca sexta must attain a threshold size before onset of the moulting cycle can occur (Truman, 1972; Truman & Riddiford, 1974; Nijhout, 1973, 1975, 1976), as in H. melpomene.

What is the adaptive significance of the existence in these species of a threshold size for moulting cycle initiation, and why does a threshold size appear not to exist in Tineola biselliella, Galleria mellonella, Ostrinia nubilalis or Tenebrio molitor ?

A possible answer lies in an examination of the food availability in the environment inhabited by each of these species. A large number of factors acting singly or in combination probably influence the rate of size increase of insect larvae, but it is considered likely that

156 S. W R I G H T A N D K . U. C L A R K E

the most important of these is food quality and availability. Tt is in food availability that the environment of H. rnelpornene and of Manduca sexta differs from that of the other species cited. The former live upon plants which have widely spaced leaves-Passijora in the case of H. melpomene and tobacco in the case of M. sexta, and it is likely that they have to travel considerable distances over the plant in order to find sufficient to eat. In addition, both species consume a large quantity of food during larval life, and are likely to strip a plant upon which they are feeding before they have completed their development- especially if there are several larvae upon one plant. Tt is also likely that they may fall from the foodplant from time to time during their development and thus face periods of food deprivation during their search for new plants and whilst climbing back up the stems. Both the larvae of H. melponiene and of M. sexfa are highly motile, and are therefore well adapted to search for new food supplies under the conditions cited above. It was observed that if larvae of H. melpomene were deprived of food, both their rate of locomotion and the amount of time spent engaged in locomotory activity greatly increased-presumably this is an adaptation enabling them to find fresh food supplies. Delay of the onset of the moulting cycle until a threshold size has been attained is probably a safeguard to ensure that the larvae of species such as H. melpoinene and M. sexfa do not metamorphose to dwarf adults during the periods of food deprivation that, as a consequence of the nature of the food eaten by these species, are likely to be quite frequent during their development. Adults of below normal size are likely to be of lower fecundity than those of normal body size and would therefore be selected against. The insect species which will undergo ecdysis in conditions of food deprivation are, in contrast to those that do not, ones which live surrounded by their food, which is therefore unlikely to be scarce. Larvae of T. biselliella live in fabric, those of G. mellonella in the wax of bees nests, those of 0. nubilalis in ears of corn, and those of T. moliror in stored grain or flour. The food of all of these species can be said to form their environment. There is, therefore, no selective pressure for development of a mechanism to prevent ecdysis and/or metamorphosis under conditions of food depriva- tion since under natural conditions food deprivation will rarely, if ever, occur. Moulting cycle initiation may then, in these species, occur at set times during larval life, or at a fixed number of hours after the previous ecdysis. In the unlikely event of such a larva facing a food shortage, the nature of their food is such that they would probably be unlikely to be able to move on to fresh supplies. In addition, the larvae of all four species have rather poorly developed powers of locomotion and would be unable to travel any great distance in the search for a new food supply, in contrast to those of H. meIpomene or M . sexfa. In such circumstances it may, therefore, be the best strategy for such larvae to metamorphose to a dwarf adult rather than to delay moulting in the chance that a new food supply might be located. This is indeed what happens if larvae of T. biscelliella, G. mellonella, 0. nubilalis, or T. rnolitor are deliberately deprived of food.

In an LD 12 : 12 photoperiod regime, with “lights on” at 0830 hrs, the critical period for ecdysone in two-day IVth instar larvae ends between 1400 and 1600 hrs. In three-day larvae, it ends somewhat earlier-between 1000 and 1200 hrs. The timing of this event was determined by conventional ligature experiments which rely on the principle of blocking the passage of a-ecdysone from the prothoracic glands to the abdomen. It is interesting that the results obtained from ligaturing IVth instar larvae of H. melpomene differ funda- mentally from those obtained by Locke (1969) and Weir (1970), working on the larvae of Culpodes e thhs . These authors produced evidence from ligation and from ultrastructural

CONTROL OF MOULTING I N H . M E L P O M E N E 157

studies that in C. ethlius the abdominal oenocytes rather than the prothoracic glands are the source of a-ecdysone. If this were also true of H. melpomene, a situation such as that observed in three-day larvae at 1000 hrs and in two-day larvae at 1200 and 1400 hrs (where the part of the body anterior to a ligature between the thorilx and abdomen formed new cuticle but that posterior to it did not) would be unlikely to arise. Weir (1970) showed that in C. erhlius such a situation did not arise-on no occasion did new cuticle form in only the anterior part of the body of a larva ligatured in this position. In Tenebrio molitor too, the abdominal oenocytes are capable of synthesizing a-ecdysone under certain experimental conditions (Romer, 1973), although whether they do so normally in this species is uncertain. It seems that these two insects may represent exceptions rather than the rule, since in other species, e.g. Bombyx mori (Fukuda, 1944), Hyalophora cecrojpia (Williams, 1952), Manduca sexra (Truman & Riddiford, 1974) and Rhodnius prolixus (W igglesworth, 1934), the results obtained from ligaturing between the thorax and the abdomen before the end of the critical period for ecdysone were similar to those obtained in H. melpomene, i.e. new cuticle formation occurred anterior to the ligature only. Moreover, it has now been demonstrated by tissue culture of isolated prothoracic glands that, at least in Manduca sexta, these glands produce large amounts of a-ecdysone (Chino, Sakurai er al., 1974; King, Bollenbacher et al., 1974). King (1972tr, b) found no evidence that in M. sexta the oenocytes are capable of producing a-ecdysone, nor that they can convert it to the P-form-although such conversion did occur in the fat body, gut, body wall and malpighian tubules.

Initiation of the moulting cycle of H. melpomene is synchronized with respect to the time of day. The mechanism by which larvae perceive signals from the photoperiod regime was not investigated-evidence from other species suggests, however, that in insects such signals may be perceived in one of two ways-either by the ocelli or directly by the brain through the transparency of the overlying cuticle. Examples of insects in which the ocelli play an important part in perceiving photoperiodic stimuli are the lepidopterans Dendrolimus pini and Aeronycta rumius (Geispits, 1957). In several blattarian species, Brousse-Gaury (1968, 1969) found that axons from the light-sensitive ocelli synapsed with the perikarya of neurosecretory cells in the tritocerebrum of the brain, and suggested that neurosecretory cell activity might be controlled by such photoneuronal pathways. Insects in which light is perceived directly by the brain include the aphid Megoura viciae (Lees, 1964), Pieris brassicae (Claret, 1966) and Anthevaea pernyi (Shakhbazov, 1961 ; Williams & Adkisson, 1964). In the latter, a transparent window is present in the area of the pupal cuticle overlying the brain, and a photosensitive pigment in the brain responds directly to light which enters through this window. When daylight increases to a critical number of hours, the brain N.S. cells respond by producing PTTH. Either of these two mechanisms may be involved in perception of signals from the photoperiod regime by H. melpomene. The ocelli are well-developed, but the cuticle of the head capsule certainly appears to be sufficiently translucent to allow light to reach the brain directly.

The gate during which moulting cycle initiation may occur appears to open at approxi- mately the time of “lights on”. It is therefore conceivable that the stimulus of “lights on” may be needed daily in order to open the gate-perhaps following the “hourglass principle”. (Lees, 1966). Examples are well known amongst both vertebrates and invertebrates, how- ever, where exposure to a photoperiodic stimulus is necessary only to synchronize an intrinsic daily rhythm in the animal so that once entrained in the absence of the photo- periodic cue the rhythm persists-free running with a periodicity of approximately 24 h.

158 S . W R I G H T A N D K . U . C L A R K E

Circadian rhythms in biological systems have been reviewed by a number of authors (amongst them Bunning, 1967 ; Cloudsley-Thompson, 1970; Danilevsky et a]., 1970 ; and Pittendrigh, 1954). Although larvae of H. melpomene do not moult each day, and cannot therefore be said to exhibit a circadian rhythm of moulting cycle initiation or of ecdysis, it is possible that each day they enter a state of physiological readiness in which to undergo moulting cycle initiation-the latter only being realized if threshold size has been exceeded.

By what mechanism might attainment of threshold size and perception of signals from the photoperiod regime be integrated to implement onset of the moulting cycle? Two possibilities seem reasonably likely, both based upon an assumption that attainment of threshold size is signalled by nerve impulses, perhaps arising from proprioreceptors sensi- tive to cuticular distension or stretching-as discussed above. The transmission of the nerve impulses which herald attainment of threshold size may be inhibited until opening of the photoperiodically controlled gate. When the gate opens, the resulting removal of this inhibition permits such impulses to trigger moulting cycle initiation. Alternatively, nerve impulses which indicate attainment of threshold size may be generated from the moment at which this size is reached, but they must be integrated within the brain with neural signals elicited by signals from the photoperiod regime during the gate. Only when signals from both sources coincide can an integrated neural signal be sent to the neuroendocrine system to trigger moulting cycle initiation. The neuroendocrine system of H . melpomene has been investigated in connection with the work presented in this paper and will be described else- where.

What is the functional significance of synchronism in the time of day at which onset of the moulting cycle occurs?

In many animal species, both vertebrate and invertebrate, the photoperiod regime provides the signal for certain stages in the life cycle to occur, or for certain events to take place. Via an effect on the hormonal system of an animal, it elicits seasonal responses to the environment such as reproductive activity, diapause or hibernation, and the adaptive colour changes seen in a number of vertebrates (see reviews by Adkisson, 1966; Cloudsley- Thompson, 1970; Danilevsky, 1965; Danilevsky, Goryshin et a/. , 1970; and De Wilde, 1962). It also elicits daily rhythms of activity, oviposition and feeding behaviour, for example (see reviews by Bunning, 1967; De Wilde, 1965; Lees, 1966 and Pittendrigh, 1960). The function of photoperiod control of both seasonal and daily activity is to ensure that the life-cycle of an animal is adapted to avoid unfavourable environmental conditions- either those which occur annually or those which occur daily for a particular species. H . nielpomene race thehiope exhibits no annual behavioural cycles such as diapause or seasonal reproductive activity, since in the tropical rainforests of the southern Amazon there are no seasons as such. There are 12 hours of light and 12 of darkness all year round, and conditions are favourable for the species throughout the year. It does, however, exhibit a daily response to photoperiod-synchronization of the time of day at which moulting cycle initiation occurs in the larvae, and consequently of the time of day of ecdysis. It seems likely that this has evolved in order to avoid unfavourable conditions for ecdysis which occur at certain times of day, since it is at this time that larvae are at their most vulnerable.

In the native habitat of H. melpomene race thelxiope, conditions of temperature and humidity vary little during the course of each day, and are very similar to those in which the experimental larvae were reared in the insectary. It is probable, therefore, that in the

CONTROL OF MOULTING I N H . M E L P O M E N E 159

wild the length of the moulting cycle of H . melportzene will be similar to that observed in the experimental larvae, and that new cuticle formation will be completed approximately 24 h after its initiation, resulting in ecdysis in the early part of the morning. Since the rain- forests surrounding Belem lie almost on the equator, dawn and dusk will occur close to 0600 and 1800 hrs respectively, and thus a three-day IVth instar larva, for example, would be expected to undergo ecdysis between 0600 and 0900 hrs. What unfavourable conditions exist in the native habitat of H . melpomene that have led to the synchronization of ecdysis in the early morning? Several possibilities can be envisaged. The larvae are likely to be at their most vulnerable to attack by predators or parasites during and shortly after ecdysis. The violent muscular contracts necessary to enable a larva to escape from its old cuticle would be likely to attract the attention of predators and, for approximately one hour following ecdysis, larvae are devoid of their array of sharp protective spikes-until those on the new cuticle are inflated. Possibly, therefore, larvae may undergo ecdysis in the early morning to avoid the attention of certain predators which are only active at other times of day. However, many predators are at their most active at the time of day at which larvae undergo ecdysis, particularly birds. If predation were the major selective pressure for synchronism of ecdysis, then ecdysis would perhaps be expected to occur later in the day rather than in the early morning. A much more likely reason for synchronization of the time of day of ecdysis in H. melpomene is as follows:

In the southern Amazonian rainforests, heavy convectional rainstorms occur regularly each day throughout the year, always in the late afternoon. If a larva was to undergo ecydsis at this time of day, such heavy rain would be likely to interfere seriously with the process. It was observed that any mechanical interference during the act of ecdysis im- paired the ability of a larva to free itself from the old cuticle and heavy rain falling on a larva could certainly be likely to produce such an effect. '4dditional hazards faced by a larva undergoing ecdysis during a rainstorm would be waterlogging and the possibility of being knocked from the foodplant by the force of the rain on the leaves. During ecdysis and the pre-ecdysial period, larvae are unable to spin a silk thread to prevent themselves from falling completely off a plant once dislodged. By undergoing ecdysis in the early morning, larvae will completely avoid these problems (cited above) and will have safely completed ecdysis before the late afternoon rainstorm.

It has been suggested that the adaptive significance of the mechanism which synchronizes moulting cycle initiation lies in the resultant synchronization of ecdysis some 24 h later. If this is the case, they why does the synchronizing mechanism act upon the beginning of the moulting cycle instead of directly upon the end of it? The explanation may be that if it were only ecdysis that was synchronized with respect to the time of day (with the moulting cycle proceeding as soon as threshold size was attained) larvae just missing the gate at the end of the moulting cycle would have to spend up to 24 h lying motionless with a slipped head capsule whilst waiting for the next gate. This would represent a considerable pro- portion of the instar, and would be rather wasteful, since the larvae would be incapable of feeding or growing during this period. In addition, they would probably be more vulnerable to attack by parasites. Since it is initiation of the moulting cycle that is gated however, larvae just missing a gate can continue to feed and grow during the period in which they are waiting for the next gate to arrive. This arrangement can perhaps only work successfully in an animal which lives in relatively constant temperature conditions and which has a rapid moulting cycle. A temperature which fluctuated unpredictably would have a

160 S . WRIGHT AND K . U. C L A R K E

desynchronizingeffect on the chemical processes which occur during a new cuticle formation. Even at constant temperature, a given degree of variation amongst individuals in the time taken for new cuticle formation would (in terms of the variation in the time of day at which ecdysis occurred) be greatly magnified in animals with a slow moulting cycle com- pared to those with a rapid one. The moulting cycle of H. melpomene is exceptionally rapid and the species lives in very constant temperature conditions in the wild-thus syn- chronization of moulting cycle initiation does result in synchronization of ecdysis. It should be found that in insects which live in more temperate regions, or which develop more slowly, any examples of synchronization of ecdysis (or of the related phenomenon of eclosion) are brought about by a mechanism which acts at a point near to the end of the moulting cycle and not upon its initiation. To date, synchronization of ecdysis has been documented in few species, although a number of instances of synchronized eclosion have been discovered, e.g. in the mosquito Anopheles gumbiue (Reiter & Jones, 1975) and in the silkmoth Anrheruea pernyi (Truman, 1971). It certainly appears that in these species a synchronizing mechanism acts at the end point of the developmental period rather than at the beginning-with animals otherwise competent to undergo eclosion waiting until an environmental cue is given before doing so.

The hormonal system mediates between the animal and its environment, adapting the timing of events in the life cycle of a species to its surroundings. Since each species is adapted to a unique environmental niche, it can be expected that release of a given hormone will be elicited by different stimuli in different species. The stimuli responsible for eliciting hormone release have been investigated in only a relatively small number of species-but from the limited information so far available this certainly seems to be the case. The release of hormones involved in the moulting cycle of insects appears to be no exception. In Rhodnius prolixus PTTH release is elicited by intake of a blood meal (Wigglesworth, 1934, 1936, 1939), in Zeugoducus depressus by a fall in the concentration of carbon dioxide inside the squash fruit in which it lives (Takaoka, 1960), in Locusta migratoria migratoroides by foregut movements associated with swallowing (Clarke & Langley, 1962, 1963a-d), and in Heliconius melpomene by the integrated stimuli of attainment of threshold size and signals from the photoperiod regime.

summary An investigation of some of the factors controlling the moulting cycle of Heliconius

melpomene was conducted. It has shown that ecdysis occurs asynchronously in either continuous light or continuous darkness, but that in an LD 12 : 12 photoperiod regime it is synchronized with respect to the time of day-and generally occurs during the early part of the photophase. Ligaturing IVth instar larvae to determine the timing of the critical periods for PTTH and ecdysone has revealed that synchronism in the time of day of ecdysis is a result of synchronism in the time of day of onset of the moulting cycle, some 24 hours earlier.

Not all of the larvae spend the same number of days in an instar-for example, some complete instar IV in two days, some in three, and some in four. This is because onset of the moulting cycle is gated, and only occurs if larvae are over a certain body size at a critical time of day. If they are below this size, they continue to feed and grow for at least a further day before the moulting cycle can be initiated. In instar IV, threshold size is

C O N T R O L O F M O U L T I N G I N H. M E L P O M E N E 161

indicated by a body weight in the region of 65-75 mg. If larvae are experimentally deprived of food to keep them below this weight, moulting cycle initiation will not occur-and only does so at the first gate following attainment of threshold size. Some evidence was obtained that the “gate” may extend over a period of several hours. Cuticular distention may be an important factor in signalling attainment of threshold size, although this has not been investigated. It seems likely that a similar gating mechanism to that described for instar IV exists in other instars too. There is no clear relationship between the number of days spent in one instar and the number of days spent in the next, however.

It has been suggested that the existence of a threshold size for moulting cycle initiation may be an adaptation to prevent metamorphosis to dwarf adults during temporary periods of food deprivation in larval life. Synchronism of moulting cycle initiation with respect to the time of day may be an adaptation to ensure that larvae do not undergo ecdysis at a time of day when environmental conditions are least favourable-that is, during the heavy convectional rainstorms that occur regularly in the late afternoon in the South American rainforests which form the native habitat of Heliconius melpoinene race thelxiope.

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