A hormone causing pupation in the blowfly calliphora ...rspb.royalsocietypublishing.org/content/royprsb/118/807/1.full.pdf · nothing was known about the actual forces which induce,

A Hormone causing Pupation in the Blowflyerythrocephala

By G ottfried F raenkel

From the Department of Zoology and Comparative Anatomy, University College,London

{Communicated by D. M. S. Watson, F.R.S.—Received November 18, 1934— Revised March 16, 1935)

[Plate 1]

Introduction

Excellent descriptions exist of the morphological and chemical changes in the metamorphosis of insects. But until the last few years practically nothing was known about the actual forces which induce, initiate, or control moulting and pupation. In growth and metamorphosis of vertebrates the dominant controlling role of hormones becomes more and more obvious. It is especially in changes which concern simultaneously the whole body that the action of hormones is involved. The metamorphosis of insects consists also of profound changes on the whole animal which manifest themselves even more abruptly than do analogous processes in vertebrates. But it was only about 10 years ago that the action of a hormone in the metamorphosis of insects was suggested for the first time by Kopec (1922). The matter was raised again by von Buddenbrock (1930, 1931) and Koller (1929), a preliminary report having appeared in Koller’s survey on hormones in invertebrates. Recently new evidence has been brought forward by Bytinski-Salz (1933) and Bodenstein (1933, a and b). The present position of the problem is given in an excellent review by Bodenstein (1933, b), and he comes to the conclusion that the action of hormones in insect metamorphosis can be shown to be very probable from several facts, even if definite proofs are still lacking. It may be mentioned here that both Koller and Bodenstein failed to recognize the important and convincing results of Kopec.

A preliminary report of the present paper appeared in ‘ Nature ’ (Fraenkel, 1934).

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2 G. Fraenkel

E xperiments a n d O bservations

P reliminary R emarks a n d T echnique A dopted

When reared at room temperature of about 20° C, the young larvae of Calliphora erythrocephala hatch from the egg about 24 hours after egg- laying, within a further 4 days they reach their maximal size and weight. In this state, which will be referred to as the mature larva,* the larva stops feeding, the crop becomes gradually emptied and reduced. This stage lasts about 3 days, then pupation starts. About 2 hours before pupation, the external appearance of the larvae becomes changed. Up to this time the skin looks shiny, it then becomes matt white. The movements gradually slacken and the shape becomes relatively shorter and broader. If the first individuals of a culture, all specimens being of the same age, are about to pupate, it can be expected with certainty that 70 to 80% of the whole batch will pupate within the following 24 hours.

The act of pupation itself is a very complicated process, consisting of several different reactions between which the connections are still entirely obscure. Roughly three reactions may be distinguished externally—

(1) the well-known barrel shape of the pupa is assumed (white pupa);(2) within a few hours the skin (cuticle) becomes hard;(3) at the same time the skin darkens.

The two latter reactions—for some reason not yet known—seem to be indissolubly coupled. Usually the conditions of the experiment prevent the use of the former reaction (1) as a criterion for pupation. In the following statements, therefore, by pupation is understood the hardening and darkening of the skin. The complicated processes of histolysis and histogenesis which follow the actual pupation are not considered in the present paper.

The changes in the larval cuticle described above, whereby thepuparium is being formed, have always been called pupation and will be referred to in this paper by this term. But it should be kept in mind that they represent really only the first inception of pupation. About 20 hours later the larval hypodermis becomes separated from the cuticle and during the second day after the formation of the puparium the real pupa is

* This stage has been called in my preliminary note (Fraenkel, 1934) prepupa in accordance to a definition given by Wardle (1930). But the very similar term prenymph has already been used by Lowne for the stage between the formation of the puparium and the formation of the pupa. The term resting larva, used by Lowne, does not adequately describe this stage, since the larvae are continuously moving about. For these reasons I adopted the term mature larva, used by Hewitt (1914).



formed by evagination and prolongation of the cephalic and thoracic imaginal discs.

One of the chief methods used was a functional separation of prepupae into two or three parts by tight ligatures of cotton or silk thread.

In the course of the work the necessity arose to inject blood of mature larvae into others. This was attended at first by great difficulties, since on piercing the cuticle a part of the body content flows out. This difficulty was finally overcome by the following arrangement: the front part of a larva was ligatured off lightly with only one knot and cut off in front of the knot. Then the blood of another larva was pressed out on a glass plate and sucked into a small glass pipette. The pipette was introduced into the larva through the knot and the fluid driven in. As the pipette was removed, the knot was tightened.

L ig a tur in g E xperiments

If old mature larvae of Calliphora erythrocephala are ligatured into two parts, four different results may arise: (1) both the anterior and posterior part pupate. This occurs only within about 16 hours after the ligature is made (at 20° C). The front part is usually but not always in advance of the hind part by 1-2 hours, fig. 1, Plate 1. (2) Only the hind part pupates within about 16 hours. This case occurred comparatively seldom (only 11 times out of many hundred larvae ligatured), fig. 2, Plate 1. (3) Only the front part pupates, the hind part remainingunpupated. This result occurs only after more than 16 hours from ligaturing, fig. 3, Plate 1. (4) Neither the anterior nor the posterior part pupates.

These facts are illustrated by a review of a great number of experiments, Table I.

Pupation in the Blowfly Calliphora erythrocephala 3

Time of pupation after ligaturing

hours 1-10

10-16 16-20 20-24 24-36 36-48 48-72 72-96

B 2

T able I

Number of specimens

Both parts pupate

176 34 12

1 ?

Anterior part alone pupates

240307181828

Posterior part alone pupates



4 G. Fraenkel

Unfortunately the experiments could not be carried out at a constant temperature, but at room temperatures of 15-20° C. In all the 12 tests, when both parts pupated after 16-20 hours, the temperature was exceptionally low (10-15° C), so that in no case did pupation of the hind part occur after more than 16 hours at 20° C.

The results of these experiments are the same whatever be the position of the ligature, provided that this be placed behind the sixth segment in the sense stated by Hewitt (1914). In actual practice this means behind the fourth of the grooves which surround the larva. If this condition be met the anterior part always contains all the ganglia which are concentrated in the fly larvae into a single mass roughly between the fifth and sixth segment, fig. 4. For convenience this ganglionic mass will be referred to as the “ ganglion,” a term introduced by Hewitt (1914).

But if the ligature is laid down in front of the fifth segment the “ ganglion ”), the hind part behaves exactly like the front part in the experiments described above, both parts pupating within about 16 hours after ligaturing or the hind part alone pupating more than 16 hours after ligaturing, figs. 5 and 6, Plate 1. The presence of the “ ganglion ” can easily be recognized

X SHeTiu)f ** 'he shape of the pupated parts. Only the g, ganglionic mass; n, Part whlch contains the ganglion becomes nerves; 1-13, body seg- properly barrel-shaped, whereas with the “ gang- ments; I, ligature in front lion ” missing pupation consists merely of hard- o f th e ganglion ; II, ening and darkening of the skin, without change ligature behind the gang- 0f t^e shape (owing to the complete lack of

nervous centres in the latter case).It seems superfluous to add that the results are exactly the same if one

of the parts is entirely cut off after ligaturing.In both the ligatured anterior and posterior parts, the development

goes on after the formation of the puparium. The hypodermis becomes separated from the cuticle. In the front part by evagination of the imaginal discs the head, thorax, and appendages of the pupa are formed, in the hind part the larval tracheal trunks are withdrawn from the abdomen. Therefore the use of the term pupation does not merely




mean formation of the puparium, but it implies formation of the pupa. These conditions are still under investigation.

From these experiments the following conclusions can be drawn.There is something in the anterior part which induces or initiates

pupation in the posterior part. About 16 hours before pupation this induction is already accomplished. Therefore a part separated from the source of the induction within this period is able to pupate. But if the ligature is made more than 16 hours before pupation, inducement to pupate cannot reach the hind part. The front part pupates if it lives long enough; this may happen even 3 days after ligaturing. But usually the anterior part, which failed to pupate, perishes after a few days, whereas the posterior part remains alive for a much longer time. In the few cases where only the posterior part pupated the ligature was laid down less than 10 hours before pupation. But an unknown factor inhibited the pupation of the front part. A suggested explanation of this peculiar behaviour is given on p. 9.

It follows with sufficient clarity from these experiments that the inducement to pupate originates either in the “ ganglion ” itself or in its immediate neighbourhood. The presence or absence of the “ ganglionic ” region determines the fate of the ligatured part.

Since the ligatured hind part contains only nerves and no ganglia, then the presence of the “ ganglion ” is not required for pupation unless it was removed more than 16 hours before.

E vidence for the A ction of a H ormone Initiating

P upation

There are two possibilities concerning the nature of the stimulus to pupate. It may consist of a nervous stimulus brought to the skin by the nervous system, or of a special hormone, secreted in the front part and carried about by the blood. In order to decide this question, the following experiments were carried out.

(1) Old mature larvae were ligatured tightly and the ligature was removed after about 1 hour. In these specimens the nervous conduction between the two parts of the body is interrupted, but the blood circulates freely between them. The interruption of the nervous conduction can be recognized easily by the external appearance of the larvae. Only the part in front of the ligature continues to move about, whereas the part behind it becomes, and remains, motionless owing to the lack of nerve centres. At the same time controls of the same age were ligatured and the ligature left until pupation. If we suppose that the induction of



6 G. Fraenkel

pupation consists of a nervous stimulus, then in all the experiments where pupation takes place more than 16 hours after ligaturing only the anterior part would pupate. The result would be the same in the specimens in which the ligature was removed as in those in which the ligature was left. But if the induction is carried out by a hormone, a different result would arise. The two parts of a larva would pupate simultaneously, if the blood circulates freely between them, even though their nervous connection is severed.

The result was unambiguous. In all specimens where nervous connection between the front and hind part was broken and the blood circulation restored, pupation occurred simultaneously in both parts of a larva. The front part never pupated alone. Altogether 34 specimens thus treated pupated more than 20 hours after ligaturing, 6 after 20-24 hours, 28 after 24-72 hours. In all these specimens the hind part would never have pupated if the ligature had been left. Of the controls, 29 pupated more than 20 hours after ligaturing and in all these only the anterior part pupated.

(2) In order to ascertain if the agents initiating pupation are carried about by the blood, injection experiments were undertaken. The technique is described on p. 3. After overcoming many difficulties, positive results were gained with two different arrangements. In all the experiments blood of larvae which were about to pupate within 2 hours was injected into posterior parts of younger ones.

(< a) Hind parts of old mature larvae which would apparently have pupated after about 16 hours were injected with “ pupation-blood.” Twenty-four hours after injection those which had not yet pupated and would hence never have pupated were injected a second time. Of 44 twice-injected specimens 18, i.e.,41%, pupated within 12 hours after the second injection. In these specimens only something contained in the injected blood can have induced pupation.

(b) Later the experiments were carried out with a simpler and more conclusive arrangement. Old mature larvae were ligatured and from 20 hours later those of them were picked out in which the anterior part alone had pupated. These posterior parts (which normally never would be able to pupate) were injected with “ pupation-blood.” The results of 12 different sets of experiments are summarized in Table II. The experiments were carried out partly at room temperature of about 20°, partly at 26° C. Group 1 consists of those in which the front part pupated within less than 28 hours, and Group 2 of those in which the front part pupated within 35-48 hours after ligaturing. One experiment in which the front parts alone pupated within 30-40 hours after ligaturing at room temperature is



included in Group 1, because at 26° C their pupation would have been expected at a much earlier point.

The result of the injections is very different in the two groups. In Group 1 of 48 injected hind parts, 22, i.e., 46%, pupated, in Group 2 of 28 injected hind parts only 2, i.e.,7%, pupated. This result indicates that pupation of the posterior parts of mature larvae can be induced by the injection of pupation blood where the stage of development of the larvae is such that pupation of the anterior half takes place within 24 hours. While if the larvae were ligatured at an earlier stage of develop-


T able II— In d u c t io n of P u patio n by Injection

Time after Number of Number of Number ofligaturing when injected pupated unpupated Number of Temperaturefront part alone hind hind hind deaths

pupated parts parts partshours

G roup 1Less than 24 10 4 3 3 RoomLess than 24 1 1 — — RoomLess than 24 2 1 — 1 Room

30-40 10 5 3 2 RoomLess than 28 9 1 5 3 26° CLess than 28 11 7 2 2 26° CLess than 24 5 3 — 2 26° C

48 22

G roup 235-48 4 *»

4 — Room35-48 . 5 — ? ? Room35-48 9 — 4 5 26° C35-48 4 1 3 — 26° C35-48 6 1 4 1 26° C

28 2

ment, such that 35-48 hours were necessary for the pupation of the anterior ends, injection of pupation blood usually failed to induce pupation of the posterior half. All pupations induced by injection occurred within less than 24 hours after injection.

From these results two conclusions may be drawn—

(1) the pupation-inducing principle becomes ineffective after somewhat less than 24 hours;

(2) in ligatured larvae the anterior part develops further towards pupation, whereas the posterior part remains in the stage at which



8 G. Fraenkel

it was ligatured. This follows from the fact that injection of pupation blood has different effects on ligatured posterior ends according to whether the front parts pupated 24 or 48 hours after ligaturing.

Since pupation can be induced by two subsequent injections, it would seem possible to develop hind parts of young mature larvae into pupae by several consecutive injections. Unfortunately this experiment cannot be carried out owing to the high death rate of the injected animals.

T he Site of O xygen U ptake d u r in g P upation

Since the discovery of tyrosinase in the blood of insects by Dewitz in 1902, the colouring of the insect skin after moulting or metamorphosis is understood as a fermentative process. A chromogen, probably tyrosin, is converted by the action of the ferment tyrosinase into melanin in the presence of molecular oxygen. The necessity of oxygen is easily shown by placing white pupae in an atmosphere of pure N 2 or H2 or C 0 2. Here they remain absolutely white for hours. Colouring starts immediately afterwards if oxygen is allowed to act upon them.

In the larval blood both chromogen and ferment are present. The blood very quickly becomes dark if exposed freely to the air. So far it is not clear how the colouring of the blood is prevented inside the body.

As to the mechanism of the colouring of the skin after moulting, it is generally believed that this process is provoked by the first exposure of the new—uncoloured—skin to the atmospheric oxygen. For the pupation of flies this view cannot be true, because the puparium is identical with the larval cuticle of the last moult. Therefore the colouring of the pupa can only be regarded as a consequence of changes inside the insect. If we pass over the nature of these changes, the question arises whether the oxygen required for the colouring process derives from the surrounding air, or from the air contained in the tracheal system.

For the solution of this question, it is a very favourable fact that in the fly larva only two parts of stigmata exist which are situated on the two ends of the body. If a white pupa is almost entirely immersed in oil, leaving only the posterior end with the hind spiracles in contact with air, the colouring and hardening occur simultaneously over the whole body at almost the same rate as normally. But if the reverse experiment is carried out, a different result occurs. At first only the part outside the oil pupates ( i.e., becomes dark and hard), then pupation spreads



gradually from the front end backward. During this process a colour gradient from the front to the hind end is visible, fig. 7, Plate 1. Finally the pupation reaches the hind end unless the animal dies before this. The colouring of the whole animal by means of the front stigmata requires about a day compared with a few hours in the normal case.

From these experiments two conclusions may be reached: (1) during pupation the oxygen necessary for the fermentative process is brought to the skin through the tracheal system; (2) the amount of air supplied through the posterior stigmata is greatly superior to that supplied through the front stigmata. This can easily be explained by the much larger size of the hind stigmata.

These conclusions are confirmed by the following experiment. When old larvae which are about to pupate are ligatured twice, into three parts, the anterior and posterior ends pupate successfully; but the middle part, the tracheal system of which is entirely cut olf from the outside air, either does not pupate at all, or becomes only very slightly coloured, fig. 8, Plate 1.

We may find in these facts the key to the peculiar case in which only the posterior part pupated (p. 3). Possibly, the anterior part is prevented from pupation by lack of oxygen.


D iscussion

The work up to the end of 1933 containing evidence for the presence of hormones causing metamorphosis in insects has been reviewed and discussed recently by Bodenstein (1933, b). It will be mentioned here only briefly so far as the present results are concerned. For the experiments of Buddenbrock (1930) the original paper must be consulted in which the results given are much less positive than in the preliminary report in Koller’s review (1929). Buddenbrock injected blood of caterpillars which were due to moult or pupate within a short time into younger •ones. These seemed to moult or pupate a little earlier than they would have done without injection. But a statistical review of the results showed that the difference was within the limits of experimental error. A much more positive result is contained in Bodenstein’s experiments in which legs of caterpillars were transplanted into older specimens of the same instar. The implant always moulted simultaneously with the host. If legs of the last instar were transplanted into younger instars, an additional moult of the transplant was induced. These results show dearly that moulting is controlled by a substance contained in the blood.



10 G. Fraenkel

The most important previous experiments to be mentioned here are those of Koped (1922). He deprived caterpillars of Lymantria dispar of the last instar of their supra-oesophageal ganglion. These caterpillars pupated if the ganglion was removed 10 days after the last moult or later, but failed to do so if the “ brain ” was removed 7 days after the last moult. The same conclusions resulted from ligaturing different parts of the body. “ The front fragments undergo metamorphosis in a normal manner, provided the caterpillar does not die of starvation, but the middle and hind fragments undergo pupation only if they have belonged to caterpillars which would have pupated in a few days.,v When the nerve cord only was cut, the whole animal pupated simultaneously. Kopec concluded from his experiments that a hormone is produced in the “ brain ” which induces pupation in the whole animal. This conclusion seems to require only a comparatively slight correction. It is not proved that the ganglion itself represents the hormone-producing, organ. Two possibilities suggest themselves, either the secretion of a gland situated elsewhere may be induced by the “ brain,” or the gland may be represented in the corpora allata which are closely connected with the “ brain.”

This was the position of the problem about six months ago. All these researches were carried out with lepidopterous larvae.

Simultaneously with my researches on the blowfly the mechanism of moulting in the tropical bug Rhodnius was investigated by Wiggles-worth (1934, a, b). Here definite proofs for the action of hormones causing moulting and metamorphosis have been established. There is a critical period, a certain time after feeding, before which moulting is prevented by decapitation, whereas bugs decapitated after this period do moult. “ If the blood from an insect decapitated after this critical period is allowed to circulate in an insect decapitated before this period the latter is caused to moult.” The source of the hormone is suggested to be the corpora allata, which undergo conspicuous changes during the critical period.

It seems, therefore, from the investigations of Kopec, WigglesWorth, and the writer that the mechanism of moulting and pupation includes a very similar if not identical principle in three different insect orders, namely, in Lepidoptera ( Lymantria dispar), Hemiptera ( and Diptera ( Calliphoraerythrocephala). At a definite period shortly before moulting or pupation a hormone is secreted in the anterior part of the body and carried about by the blood. Separated posterior parts only moult or pupate if the separation took place after this hormone was released and discharged. Disconnection of nervous transmission does



not prevent the hind part pupating if the blood circulation is maintained (.Lymantriaand Calliphora). Posterior parts separated before this period, which otherwise would not pupate, can be induced to pupate or moult by the injection of blood of larvae which have already passed this period ( Rhodnius, Calliphora).

In Lymantria and Rhodnius the hormone is produced in the head, possibly in the corpora allata. In the headless fly-larva the source of the hormone was localized in the anterior part of the body in the immediate neighbourhood of the “ ganglion.” If we could assume that the “ ganglion ” itself produces the hormone, Kopec’s as well as my results would be satisfactorily solved. In the light of the new findings on “ neurohumoralism ” and secretory function in the brain of vertebrates, the metamorphosis of insects would form new, interesting evidence. But as long as proofs are lacking—it is stated above that Kopec’s results are not conclusive—the immediate neighbourhood of the “ ganglion ” in the fly-larva has to be searched for the hormone-producing organ. There exists no organ in the fly-larva which can be homologized with the corpora allata in other insects. The localization of the pupation-gland is the object of further investigation.

I take this opportunity of thanking the Academic Assistance Council for providing a grant, and Professor D. M. S. Watson for his continual interest and his hospitality in extending to me the facilities of his department.

Summary

Proofs have been established for the action of a hormone inducing pupation in the blowfly. This hormone is secreted from 16 hours before pupation, at 20° C. The hormone-producing organ is either the “ ganglion ” or in its immediate neighbourhood. After the hormone has already been discharged, pupation can be successfully accomplished without the co-operation of the nervous centres (ganglion).

The atmospheric oxygen required for the darkening of the pupa is brought to the skin through the tracheal system.

R eferences

Bodenstein, D. (1933, a). ‘ Roux. Arch. EntwMech. Org.,’ vol. 128, p. 564.------(1933, b). ‘ Naturwiss.,’ vol. 21, p. 861.Buddenbrock, W. von (1930). ‘ Z. Morphol. Okol.,’ vol. 18, p. 701.------(1931). ‘ Z. vergl. Physiol.,’ vol. 14, p. 415.Bytinski-Salz, H. (1933). ‘ Roux. Arch. EntwMech. Org.,’ vol. 129, p. 356.




12 Pupation in the Blowfly Calliphora erythrocephala

Dewitz, J. (1902). ‘ C. R. Soc. Biol. Paris,’ vol. 54, p. 44.------(1924). ‘ Zool. Jahrb. Abt. Allg. Zool. Ph}'s.,’ vol. 41, p. 245.Fraenkel, G. (1934). ‘ Nature,’ vol. 133, p. 834, June 2.Hewitt, C. G. (1914). “ The House-Fly,” Cambridge.Hopp, M. (1933). ‘ Zool. Jahrb. Abt. Anat. Ont.,’ vol. 57, p. 433.Roller, G. (1929). ‘ Biol. Rev.,’ vol. 4, p. 269.Koped, S. (1922). ‘ Biol. Bull.,’ vol. 42, p. 323.------(1924). ‘ Biol. Bull.,’ vol. 46, p. 1.Lowne, B. T. (1890-92). ‘ The Blow-Fly,’ vol. 1, London.Wardle (1930). ‘ Ann. app. Biol.,’ vol. 17, p. 568.Wigglesworth, V. B. (1934, a). ‘ Nature,’ vol. 133, p. 725.------(1934, b). ‘ Quart. J. Micr. Sci.,’ vol. 77, p. 191.

Explanation of P late 1

All the animals are orientated with the front end towards the top of the page.Fig. 1—Both parts pupate within 16 hours after ligaturing.Fig. 2—The hind part alone pupates within 16 hours after ligaturing.F ig . 3—The front part alone pupates after more than 16 hours from ligaturing. F igs. 5 and 6—Ligature laid down in front of the ganglion.Fig. 5—Both parts pupate within 16 hours after ligaturing.Fig. 6—Hind part alone pupates more than 16 hours after ligaturing.F ig . 7—The whole body except the very front end immersed in oil during the colouring

of the pupa: colouring starts on the front end and spreads gradually backward. F ig . 8—Middle part of a twice-ligatured mature lava remains unpupated.



Fraenkel Proc. Roy. Soc., B, vol. 118, Plate 1

F ig. 1

Fig. 7 Fig. 8

{Facing p. 12)



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A hormone causing pupation in the blowfly calliphora ...rspb.royalsocietypublishing.org/content/royprsb/118/807/1.full.pdf · nothing was known about the actual forces which induce,