Exp. Biol (1962), 39, 395-4H 395

ih 1 plate and 6 text-figures

Printed in Great Britain

THE IMAGINAL ECDYSIS OF BLOWFLIES. THE CONTROLOF CUTICUAR HARDENING AND DARKENING

BY C. B. COTTRELL*

Department of Zoology, University of Cambridge

(Received 1 February 1962)

INTRODUCTION

There is evidence that the initiation of normal hardening and darkening in the blow-fly is under nervous control (Fraenkel, 1935). Flies forced to keep digging throughsawdust (or some similar medium) from the moment of their emergence may delayexpansion, hardening and darkening for periods several times greater than thosenormally required for the completion of these processes. On the contrary, if they arelightly anaesthetized with ether before being placed in the sawdust, there is no delay.

Since the epidermal cells of insects are considered to lack innervation it seemsimprobable that the control of cuticular hardening and darkening can be direct. Themost likely alternative is that it is achieved by means of a blood-borne factor the releaseof which is nervously controlled. Such a factor might function either by activatingthe reaction systems concerned, in which case initiation would be by its release, oralternatively it might be inhibitory, its presence preventing hardening and darkening.In this case initiation would result from the cessation of its production.

Using ligatures, Fraenkel attempted to eliminate a centre which might control orinitiate colouring, but his results were very inconclusive. Separated parts oftendiffered in coloration but identically treated specimens behaved differently and evenwithin one part the coloration was not evenly distributed. Coloration of all parts wasinvariably normal only in flies which had been ligatured at the time of wing expansionor shortly before. Fraenkel's experiments were repeated by the present author withequally inconclusive results and it was therefore decided to attempt a demonstrationby means of a blood-transfusion technique. The results of these experiments formthe basis of this paper. A detailed description of the imaginal ecdysis of blowflies isgiven elsewhere (Cottrell, 1962a).

MATERIAL AND METHODS

CalHphora erythrocephala (Meigen) was used throughout this study, the methods ofrearing and handling being identical to those previously described (Cottrell, 1962 a).

Transfusions were made by means of a fine-drawn glass needle (Text-fig. 1, iV)joined by a short connecting tube (C) to an 'Agla' micrometer syringe (A). The syringeassembly was clamped in a horizontal position and the connecting tube arranged sothat the needle pointed almost vertically downwards into a Buchner funnel (B)mounted on the stage of a dissecting microscope (Z)). The syringe and connecting tubeswere rilled with liquid paraffin coloured with Sudan III so that only a small air space

• Now at Department of Zoology, University College of Rhodesia and Nyasaland.

396 C. B. COTTRELL

intervened between the paraffin and the fluid to be injected. Glass needles weresealed into the connecting tube with wax-resin mixture and were thus readily re-placeable. They were 6-7 cm. long and tapered at their ends to a point 25^75 /x indiameter. Generally they were calibrated by means of a second 'Agla' syringe so as todeliver 5/xl. of fluid. Prior to injection, the newly emerged fly was suspended from afine entomological pin waxed to the mesoscutellum and embedded in a matchstick andwas then anaesthetized by a 2 min. exposure to carbon dioxide. On removal from thegas chamber, it was brought into the Buchner funnel (through which carbon dioxidewas being passed) and by means of a specially sharpened, fine steel entomological pina very small hole was punctured in the mesoscutum. The injection needle was in-serted by bringing the fly up to the needle and not vice versa. After injection thewound was sealed with wax.

Text-fig. 1. Apparatus used for injecting newly emerged flies. For further details gee text.

Blood for transfusion was obtained by pricking the ptilina of newly emerged fliesor the thoraxes of expanded ones, the drop so produced being touched to a siliconedslide. In order not to clog the fine tip of the injection needle with the small particlesof larval fat body which occur free in the blood of newly emerged flies, the blood wasgenerally ' filtered' by placing a small square consisting of two or three layers of lenstissue on top of the drop and then drawing it through into a pipette held above thetissue. The 'filtrate' was then expelled on to a clean part of the slide and the requiredamount (usually 5 /A.) drawn into the injection needle. With a little practice the wholeoperation could be accomplished in a little under a minute and subsequent injectioninto an already anaesthetized recipient occupied only about another minute.

Decapitation and other operations were carried out under carbon dioxide anaesthesia,usually over a Buchner funnel (Williams, 1946). Decapitation was performed by meansof curved irredectomy scissors, the cut being made in such a way that a very small flapof the occiput was left on the torso. This flap immediately folded down over

Imagined ecdysis and cutictdar control of blowflies 397

severed neck and greatly facilitated the subsequent sealing of the wound which wasdone with a 1:1 beeswax-resin mixture applied by means of an electrically heatedcautery. When it was necessary to remove or isolate the abdomen, a silk ligature wastied about the base before the appropriate cut was made.

RESULTS

(1) Transfusions into newly emerged flies

The first series of experiments was designed to test whether, in conditions un-suitable for expansion, hardening and darkening were being prevented by the presenceof an inhibitory factor in the blood. Blood for transfusion was freshly drawn fromdonors either at the moment of their emergence or after they had been kept digging forabout 1 hr. Recipients were anaesthetized within a few seconds of emergence by a2 min. exposure to carbon dioxide. After injection they were given the opportunityto pick up cotton-wool balls and the ability to do so was taken to indicate theirrecovery from anaesthesia. The times taken from this point to the moment of fullwing extension and from wing extension to the first signs of darkening were noted.The means for the two groups and for a third control group which was only exposedto carbon dioxide are given in Table 1.

Table 1. Mean times (in minutes) taken to expansion and to darkening by newly emergedCalliphora transfused with blood from newly emerged or digging donors (21-5 ° C.)

Minutes from Minutes fromrecovery to recovery to

No. of expansion, darkeningDonors recipients Mean±s.E. Mean±s.E.

At emergence 10 45'7±i-7 32-0 ± I - IAfter 1 hr. digging 8 41-312-0 31-411-3None (recipients given COj only) 9 44-2 ± 2-1 30-0+1-4

It can be seen that there is very little difference between the three groups. Ifhardening and darkening were being prevented by the secretion of an inhibitoryfactor in digging flies it might be expected that expansion or darkening or both wouldbe relatively delayed in those flies receiving 'digging' blood. No such effect isrecognizable.

Comparison with data obtained from untreated flies (see figs. 3 a and 3 b in Cottrell,1962a) shows that even allowing for the recovery period, exposure to carbon dioxidehas considerably lengthened the time to wing extension, although the time from wingextension to the first signs of darkening is not similarly affected. Evidently carbondioxide causes delay in the initiation of expansion.

In a second series of experiments one group of recipients received blood from newlyemerged donors, the other from flies which had been allowed to expand and from whichblood was drawn between 7 and 60 min. after the appearance of the first signs ofdarkening. As in the previous series, the group of six flies which received newlyemerged blood expanded in a normal manner (PI. 1, fig. 1 b). On the other hand, theten flies which received blood from darkening donors all hardened and darkened in an

^mperfectly expanded condition (PI. 1, fig. ia). Darkening appeared earlier than was

39^ C. B. COTTRELL

expected and it was clear that considerable hardening had occurred before sufficientair had been swallowed to expand the folded appendages. In contrast to the darkening,however, the first appearance of air-pumping movements was not earlier than in thecontrol flies and as more and more air was taken in, the membranous parts of thecuticle became progressively more distended and, due to separation of their mem-branes, the still folded wings became converted into bladders filled with blood and air.In some cases the internal pressure combined with stroking movements of the legswas sufficient to split these bladders but in no case did any appreciable expansion ofthe presumptive sclerotized cuticle occur.

This experiment, taken together with the previous one, provides clear evidencethat:

(1) Some factor is present in the blood of darkening flies which is necessary forhardening and darkening.

(2) This factor is not present in the blood of digging flies.(3) The cuticle is competent to undergo hardening and darkening before the fly

has expanded or reached a state at which it would normally be expected to expand.

Table 2. Mean times (in minutes) taken by newly emerged flies transfused with

blood from newly emerged or darkening donors (260 C.)

Emergence or Emergence orNo. of Injection to recovery to Expansion to injection to

Donors recipients recovery expansion darkening darkening

Newly emerged 7 6-6 55-3±3-3 23-3 ±2-2 78-6 ±3-8Darkening flies 5 — — — 44-0 ±3-1None (recipients 10 — 2S'7±2-4 a i -a±i - 5 46-9±3-ountreated

To obtain more accurate timing the experiment was repeated and the results aresummarized in Table 2. Once again all the flies receiving blood from darkeningindividuals failed to expand while those receiving blood from newly emerged oneswere unaffected. It seems reasonable to assume that the factor present in the bloodof darkening flies is one of the links in the chain, whereby the control of hardeningand darkening is effected and, if this is so, it is of interest to try to estimate when theseprocesses are initiated. For this purpose the mean time taken from injection to theappearance of the first signs of darkening in flies receiving ' active' blood should givean indication of how long the factor requires to produce a visible effect. Table 2 showsthis ' lag period' to be about 44-0 min. but it must be remembered that in this case thereaction has been initiated by a dose of active blood which has been diluted about fourtimes by the inactive blood of the recipient. Probably the time lag would be shorter inthe intact animal receiving its own full dose. If this value of 44-0 min. is subtractedfrom the mean time taken from emergence to the first signs of darkening by untreatedcontrols (46-9) we find that these controls must have secreted their own darkeningfactor only 2-9 min. after being waxed to pins and given cotton-wool balls. For thereasons given above this value is probably an underestimate, but whatever the trueone, it is certain that the processes of hardening and darkening are initiated very soonafter the fly reaches conditions suitable for expansion and certainly long beforeexpansion has been completed.

Imaginal ecdysis and cuticular control of blowflies 399

Assuming that darkening becomes visible some 44/0 min. after the release of thedarkening factor, subtraction of this figure from the mean time taken from injectionto darkening by flies receiving 'inactive' (i.e. newly emerged) blood should indicatehow long after the injection these flies secreted their own factor. This is found to be78-6-44-0 = 34-6 min. Thus the effect of exposure to carbon dioxide is to delay theinitiation of expansion and darkening for some 35 min. after the injection. Of thisperiod an average of 6-6 min. passes before the fly is capable of holding a cotton-woolball and the remainder must represent a recovery period during which no externalsymptoms are visible. Presumably the flies receiving active blood behave similarly sothat it appears that the foreign darkening factor has been introduced some 35 min.earlier than these flies would have released their own secretions. This means that thefirst signs of darkening have appeared only about 5 min. after air-swallowing and,by comparison with flies receiving inactive blood, some 11-5 min. (55-3-44-0) beforethe wings would normally have expanded. It is evident that anticipating the naturalrelease of the darkening factor by some 35 min. is sufficient to prevent the normalcompletion of the ecdysis.

Table 3. Transfusion of newly emerged flies with blood from donorsof various ages

Age of donor(minutes Effect on recipient

after , * >emergence) Expansion Darkening

o Normal Normal3 Normal Normal5 Slight Normal

15 Slight Normal20 Prevented Early23 Prevented Early31 Prevented Early33 Prevented Early46 Prevented Early48 Prevented Early

To check the accuracy of the estimated time of release of the darkening factor, aseries of newly emerged flies was transfused with blood from donors of various ages.The results are given in Table 3 and seem to show that the blood-borne factor isreleased between 3 and 15 min. after emergence. This is in approximate agreementwith the estimated figure of 2-9 min. arrived at above. Experiments involving thetransfusing of blood into newly emerged individuals are very time-consuming and thematter was not pursued further. However, additional estimates of the ' critical period'for the release of the darkening factor into the blood have been obtained by othermethods (see below and Cottrell 1962 b) and are found to agree with the results givenin Table 3.

(2) Transfusion into digging flies

Although the transfusion of blood into newly emerged flies seems to show thatdarkening is initiated by the release of a factor into the blood, it is still possible toenvisage some more complex system where additional inhibitory factors might also

^ e involved in the [delay during [digging. To [investigate this possibility and more2fi Exp. BioL 39, 3

Receivingnewly

emergedblood

13

0

31 0

0

49

Darkeningblood

14

140

0

140

0

4-OO C. B . COTTRELL

particularly to s tudy the relationship between air-pumping and darkening, blood wastransfused into digging flies. T h e experimental set u p was as follows: on emergenceflies were set to dig in capillary tubes. After i hr., any which showed signs of darkeningwere rejected, the remainder served as recipients. While still within their tubes theywere anaesthetized with carbon dioxide, removed, transfused with blood and replacedin tubes to recover from anaesthesia. They were kept in the tubes for a further 2 hr.and were then removed, waxed to pins, given cotton-wool balls and kept under obser-vation for another 3 hr . One group of flies received blood from newly emerged donors,the other from darkening ones. As it was seldom possible to do more than one or twopairs in a day, the experiments were not all performed at the same temperatures, thevariation being from about 20 to 25 ° C.

Table 4. Effect of transfusion on digging flies

Total number of flies...

,. . . f(i) Completely dark: final removal I ( 2 ) s ^ h t l d a r k

from diggmg tubes ^ Q ^ p^Ability to expand after f(i) No expansion

final removal from digging-! (a) Partial expansiontubes 1(3) Complete expansion

The results are given in Table 4. They show that when the donor is a newlyemerged fly, the recipients are usually little affected (PL 1, fig. 2 a, b). They continueto dig for the full 2 hr. after the injection and most individuals subsequently expandand darken in a normal manner (PL 1, fig. 3 a, b). Four of the thirteen flies failed toexpand completely, but comparison with data for untreated flies (Table 4, Cottrell,1962a) indicates that this is not an abnormal proportion after a total digging period of3 hr. Of these four flies, three showed slight patchy abdominal darkening when theywere removed from the tubes and this would appear to be a form of the ' secondarydarkening' discussed elsewhere (Cottrell, 1962a). The other ten flies were com-pletely pale when they were removed.

The result was quite different when the donor was a darkening fly. All of thefourteen recipients of 'active' blood darkened uniformly and completely while stillwithin their tubes (PL 1, fig. 4a, b). The first signs of darkening were apparent between42 and 65 min. after injection (mean ± s.D. = 48-4 ± 6-8 min.) which agrees reasonablywell with the lag period of 44-o + 3a min. obtained by transfusion into newly emergedflies at 260 C. The darkening was not accompanied by any attempt at air-pumping(i.e. expansion) and as it progressed it increasingly interfered with the developmentof normal digging movements. Nevertheless, these movements continued in a modifiedform until the flies were removed from their capillary tubes.

After hardening in such cramped conditions, the flies were generally incapableeither of holding on to cotton-wool balls or of reaching their ptilina with their fore-legs. In spite of this most specimens began to swallow air some 10-20 min. afterremoval and they then went through a cycle of air-pumping which differed only slightrji

Imagined ecdysis and cuticular control of blowflies 401

from the normal one. This resulted in extreme distension of all membranous areasand in the conversion of the unexpanded wings into sacs filled with blood and air (seePI. 1, fig. 5 a, b) but not, of course, in any normal expansion.

Transfusion of active and inactive blood into digging flies completely confirmsexperiments with newly emerged ones and (at least in so far as the haemolymph isconcerned) gives no indication that more than one factor is involved in the control ofhardening and darkening. The experiment also indicates that the presence in the bloodof the active factor responsible for darkening is not the immediate cause of theinitiation of air-pumping.

Table 5 gives a summary of the results of all the transfusion experiments.

Table 5. Summary of transfusion experiments

Recipient

Newly emerged

Treatment ofrecipient after injection

Placed in conditions suitablefor expansion

Digging for I hour Kept digging for a hr. afterinjection then placed inconditions suitable forexpansion

Donor

Newly emerged

Digging i hour

Darkening

Newly emergedDarkening

Result

Expansion: normalDarkening: normalExpansion: normalDarkening: normalExpansion: prevented (butair-pumping normal)darkening: slightly earlierthan normal

Expansion: normalDarkening: normalDarkening appears infly about 50 min. after in-jection. No air-pumpingduring digging. Air-pump-ing starts on removal butexpansion is prevented bypremature hardening ofcuticle

D f>it turn (3) Decapitation and isolation of parts

Decapitated Calliphora kept at room temperature live for 3-4 days which is as longas do flies deprived of food and water (cf. Fraenkel, 1940).

The effect of beheading twenty-five flies within a few seconds of emergence isshown in Text-fig. 2. After decapitation they were kept in a damp box at roomtemperature and the extent of their darkening was estimated at intervals, accordingto an arbitrary scale in which o represented a completely pale individual and 4 a fullydarkened one. The sum of these figures gave the ' Total Darkening Score' and aspreviously described (Cottrell, 1962a) the difference between the curve so obtainedand the curve for the percentage of flies showing some darkening gives an inverseindication of the extent of darkening shown by an average fly.

Most of the flies (22/25) showed no reaction within the first 12 hr., but when nextexamined at 24 hr. all showed some signs of darkening. This was uneven and patchyand was more extensive on the abdomen than the thorax. During the next 12 hr. itgradually increased in extent so that the whole body was dark 48 hr. after emergence.Of the three individuals which showed darkening before 12 hr. one darkened rapidly

j n d completely between 1 and 2 hr. suggesting that the darkening factor had already26-2

4-O2 C. B. COTTRELL

been secreted, while the other two showed slight darkening between 2 and 3 hr., thenremained relatively steady until 12 hr. The flies began to die between 48 and 72 hr.and all were dead by 120 hr. after decapitation.

The over-all reaction is very similar to that shown by flies which have been forced tokeep digging for long periods after emergence (see Fig. 4, Cottrell, 1962a) and itseems reasonable to assume that the slow, patchy darkening which appeared after12 hr. and which develops over the next 24 hr. is closely related to, if not identical with,the 'secondary darkening' reaction which is encountered in digging flies.

However, in the decapitated flies, darkening was much more intense and all indi-viduals were fully dark at 32 hr., long before any had begun to die. In contrast, manyof the digging flies were still not completely dark at the time of their deaths.

50 60 70 80Hours after decapitation

Text-fig. 2. Effect on subsequent darkening of decapitating CalUphora atemergence and storing at room temperature.

The activity of the tyrosinase system is known to be increased by injury (cf.Sussman, 1949) so that it seemed possible that the apparent re-inforcement of thesecondary darkening effect might be due to the tissue damage consequent on theoperation. It is widely known that post-operative shock effects may be lessened bykeeping the experimental animals at low temperatures subsequent to the operation.For instance, Van der Kloot & Williams (1954) found that spinning in the cecropiasilkworm was blocked by very trivial brain damage but that this effect was not specificand was due to 'shock': it could be overcome by keeping the caterpillars at 50 C. for3 days following the operation.

Twenty-five flies were therefore decapitated at emergence, stored in a refrigeratorat about 6° C. and their darkening was followed as in the previous experiment. Theresults are shown in Text-fig. 3. It is evident that not only did the low temperaturebring about a considerable decrease in the rate of darkening but that the extent of thedarkening reaction was also considerably suppressed. At the time of death, 16% of theflies showed no darkening at all while most were only darkened to an extent equivalentto 2 on the arbitrary scale. This means that the reaction of decapitated flies kept in therefrigerator (Text-fig. 3) resembles that of digging flies (Text-fig. 4, Cottrell, 1962 a)much more closely than it does that of decapitated flies kept at room temperature(Text-fig. 2).

Also of interest in the present experiment is the comparatively close correlation^

Imaginal ecdysis and cuticular control of blowflies 403

between the appearance of darkening and the death of the flies, an impression which isconfirmed by most of the individual records. A duplicate pair of experiments providedessentially similar results to those described here.

Whatever the mechanism, it seems clear that low temperature has a differentialeffect on what may perhaps be loosely termed ' the recovery processes' as against thoseleading to the appearance of ' secondary darkening' and that this effect is in favour ofthe 'recovery processes'. That the effect is not due simply to the suppression by lowtemperature of the activity of the enzyme producing the chromogen will appear fromsubsequent experiments.

100

80

60

40

20

—x—x—x—x-x-x-x_

°/0 survival

% of individualsshowing some

darkening

2 4 6 8 10 12 14 16 18 20 22 24Days after decapitation

Text-fig. 3. Effect on subsequent darkening of decapitating CalUphoraat emergence and storing at about 6° C.

The critical period for decapitation

To investigate the effect of decapitation at varying times, groups of ten flies werebeheaded either at the moment of emergence or after they had been allowed to remainin conditions suitable for expansion for 3, 5, 7, 10, 13 or 15 min. The bodies were keptat room temperature (180 C.) and the extent of darkening was estimated at intervalsaccording to an arbitrary scale ranging from o to 4, the individual results beingsummed to give the 'Total Extent of Darkening'. The results are shown in Text-fig. 4.

Flies decapitated either at emergence or after a 3 min. interval behaved substantiallyas in the previous experiments. There was no sign of darkening for at least 18 hr., thenbetween 20 and 29 hr. after emergence, a patchy, uneven darkening appeared andduring the next 16-26 hr. gradually increased in extent and intensity until it coveredthe whole body. As has already been pointed out, this reaction is strongly reminiscentof the 'secondary darkening' shown by digging flies and may possibly be identicalto it.

When decapitation is performed on flies which have been allowed to remain inconditions suitable for expansion for 15 min. from the time of their emergence, a rapid,even darkening occurs. This begins about 55 min. after decapitation (room temperature,180 C.) and is apparently complete about 100 min. later. It is evident that this reactionis very similar to, and probably identical with, the normal rapid darkening whichoccurs in intact flies.

As is shown in Text-fig. 4, batches of flies in which decapitation has been delayed for^ - 1 3 min. after emergence react in an intermediate manner. The longer the time

4°4 C. B. COTTRELL

elapsing before decapitation, the greater is the proportion which react in the normal rapidmanner. These results show clearly that: (i) normal rapid hardening and darkeningcannot be initiated in the absence of the head (which contains the brain and suboeso-phageal ganglion); (2) after flies have remained in condition suitable for expansion for

Decapitated 15 mln. after emergence

0

Text

4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 SO 52 54 56Time after decapitation (hr.)

:-fig. 4. Darkening of groups of ten CalUphora kept at i8° C. for various times afteremergence then anaesthetized, decapitated and stored at room temperature.

40Decapitated 15 min. after emergence

8 103 4 5 6 7Time after decapitation (days)

Text-fig. 5. Darkening of groups of ten CalUphora kept at i8° C. for various times afteremergence then anaesthetized, decapitated and stored at about 6° C.

a ' critical period' (which varies from fly to fly but lies between 5 and 13 min. at 18° C.)the presence of the brain is no longer necessary for the initiation of hardening anddarkening.

Imaginal ecdysis and cuticular control of blowflies 405

This ' critical period' corresponds to the appearance of the active factor in the bloodand it seems reasonable to infer that it is the brain and/or suboesophageal ganglionwhich in response to the external stimulus situation controls the release of the darkeningfactor. It should be noted that decapitation does not remove the ring gland (fusedcorpora allata, corpora cardiaca and degenerating peritracheal glands) which remainsintact in the thorax.

Text-fig. 5 shows the results of a repetition of the same experiment. In this casethe intervals before decapitation were o, 3, 7, 11, and 15 min. at a temperature of200 C. and after the operation the bodies were stored in a refrigerator at about 6° C.The resulting depression of 'secondary darkening' serves to emphasize the primaryeffect.

40 r

3 25

£ 2 0

15

10

20° C.

18° C.

i ' / / , 1 i 1 1 1 1 I 1 1 i I I I I I I—I2 ' 4 6 8 10 12 14 16 18

Time of anaesthetization (min. after emergence)20

Text-fig. 6. Plot of certain data from Text-fig. 4 (crosses) and Text-fig. 5 (circles) showingrelation between time of anaesthetization (i.e. decapitation) and extent of darkening. Darkeningestimated at 10 hr. for groups of flies kept at 18° C. before decapitation and stored at roomtemperature (Text-fig. 4) and 1 day for groups of flies kept at 20° C. before decapitation andstored at about 6° C. (Text-fig. 5).

Text-fig. 6 is a plot of the' extent of darkening' reached by the different batches in thetwo previous experiments after 10 hr. and 1 day respectively. Besides giving a clearpicture of the critical period it shows that the degree of darkening reached at thesetimes is at least roughly proportional to the interval elapsing between emergence anddecapitation. This suggests that the extent of darkening might be proportional withincertain limits to the amount of darkening factor which has been released into theblood.

Isolation of abdomina

The reactions of isolated abdomina are essentially similar to those of decapitatedflies. If the abdomina are isolated less than 15 min. after emergence, the darkening ispatchy, irregular and slow, becoming complete only after a lapse of some 50-60 hr.However, the situation is greatly complicated by the appearance of small dark patches

406 C. B. COTTRELL

in the neighbourhood of the ligatures. These are generally found where the cuticle hasbeen gathered into folds and in some specimens may appear as early as 6 hr. afterisolation. Later they gradually increase in extent and intensity while (as in decapitatedflies) other spots appear in the vicinity of the dorsal midline. A few specimens mayremain completely pale for almost 50 hx. but the over-all effect of the' damage reactions'resulting from severing and ligaturing the abdomina, is to advance the onset ofsecondary darkening to within about 5 hr. of isolation, without however greatlyaffecting the time of its completion.

Removal of abdomina at emergence

Since the 'secondary darkening' reaction is almost always more intense in theabdomen than in the thorax, it was of interest to see what effect removal of the abdomenhad on the behaviour of newly emerged flies. The operation was performed undercarbon dioxide anaesthesia and control groups were either untreated or were onlyexposed to carbon dioxide.

Table 6. Effect of removing abdomina at emerging {times in minutes)

Treatment

Abdomen removedCO, onlyNo treatment

Number offlies

91 0

1 0

Minutes torecovery

(mean±s.E.)

8-6 ± 0 87-2 ±o-S

—

Minutes fromrecovery or

emergence toexpansion

(mean±s.E.)

54-8 ±2-847-8 ±2-438-4±i-5

Minutes fromexpansion to

darkening(mean±s.E.)

7 I - I ± 5 - I34-3 ±o-83 i - 4 ± i o

The results are shown in Table 6. Surprisingly, all the operated flies were able toextend their wings to a considerable extent and one specimen actually managed tostraighten them completely. Even when expansion was incomplete, there was alwaysa more or less distinct point at which the wing membranes began to unfold and thiswas taken to indicate expansion. The table shows that although treatment with carbondioxide significantly increases expansion time (P < o-oi), the mean times taken fromrecovery to expansion by operated and carbon dioxide treated insects are not greatlydifferent (P < o-i > 0-05) considering the severity of the operation. Apparentlyremoval of the abdomen has no effect on the initiation of the processes leading toexpansion. The partial failure of expansion is almost certainly due to the loss of a verylarge part of the total blood volume, so that little blood is available to expand theappendages, as well as to the absence of the abdominal muscles.

On the other hand the mean time from expansion to the appearance of darkening wassignificantly longer (P < o-oi) for operated flies than for carbon dioxide treated ones.However, since darkening did eventually occur, the biological significance of thedifference is unclear; it might well be due simply to restricted circulation. Certainly,there can be no question that any component of the hardening and darkening system islocated only in the abdomen.

Imaginal ecdysis and cuticular control of blowflies 407

(4) Secondary darkening as a damage reaction

The increase in the intensity of the patchy 'secondary darkening' seen in decapi-tated flies and isolated abdomina over that in digging intact flies has been attributedto damage reactions consequent on the operation involved. The present sectionprovides further evidence in support of this view.

Table 7. Darkening in Calliphora decapitated at emergence and subsequentlydamaged by pinching their abdomina toith forceps (max. score = 15)

Manner of pinching abdomen

Longitudinal dorsal strip of cuticleLongitudinal ventral strip of cuticleLongitudinal lateral strip of cuticleTip of abdomenWhole of abdomen transversely at geg. 4Whole of abdomen compressed laterallyWhole of abdomen depressed dorso-ventrallyControls: not pinched

Table 8. Effect of crushing internal organs of decapitated Calliphora by means of forcepsinserted laterally through a slit in their abdomina (max. score = 15)

Darkening score

ahr.

i3*2

31 2 *

9*8*i

Darkening6hr.

2 *

4*2 *

8*H i12

1 3 *2

score26 hr.

1 211

2 *1 2 *

IS15156

4 hr. 10 hr. 22 hr.

Forceps inserted: internal organs squeezed 9! 9} 10Controls: forceps inserted only i * 2*

Flies decapitated at emergence and subsequently kept in the refrigerator for one daywere allowed to come to room temperature. Any which showed signs of darkeningwere rejected and the remainder divided into batches of five, the abdomina of themembers of each group being squeezed in a particular manner by means of fine watch-makers' forceps. The bodies were then kept at room temperature and the extent ofdarkening estimated according to an arbitrary scale from o to 3. The results are givenin Table 7. Pinching and crushing superficial tissues such as longitudinal, dorsal,ventral and lateral strips of cuticle (including in the case of the dorsal strip, damage tothe heart and pericardial cells) resulted in relatively little increase in secondarydarkening. However pinching the abdomen in such a manner that internal organs werealso crushed produced an impressive increase both in its rate of development and itsfinal extent. Apparently secondary darkening is produced more readily by damage tothe internal organs (most probably the gut) than to the body wall.

Further evidence is provided by the next experiment. Small slits were madelaterally in the abdomina of two batches of five decapitated flies which had previouslybeen anaesthetized with COS. In each of the first five a pair of fine-pointed watch-makers' forceps was introduced into the abdomen and then withdrawn the pointsbeing held together throughout the operation. The slits were then sealed with wax-resin mixture. In the remaining five the forceps were inserted with the points wideapart. While within the abdomen the forceps were closed, crushing any tissues

J^nainly gut and malpighian tubules) lying between them. The points were then allowed

408 C. B. COTTRELL

to spring apart, the forceps withdrawn and the slit sealed as before. The subsequentprogress of secondary darkening is summarized in Table 8 which clearly shows thatcrushing the internal organs was effective in producing secondary darkening.

Further experiments were made on digging flies. These were anaesthetized at themoment of emergence by a i min. exposure to CO8, the requisite operation was per-formed, and then the flies were returned to cotton-wool plugged tubes to recover andbegin digging. Table 9 shows that pinching the abdomen transversely across its base(in this way simulating the placing and removal of a ligature) was highly effective incausing darkening. Unfortunately the interpretation of this result is not clear-cutsince, possibly as a result of the paralysis of the abdominal muscles and the consequentabsence of normal digging movements, about half the flies were found to haveswallowed small quantities of air. Thus some of the observed darkening may have beendue to the release of the normal darkening factor. In spite of this there can be littledoubt that crushing the abdomen so as to cause internal damage does increasesecondary darkening in digging flies. On the other hand, as in decapitated flies,crushing other superficial parts such as the tip of the abdomen, a median dorsal stripof the abdominal cuticle, a median dorsal strip of the thoracic cuticle or the ptilinumhas a scarcely distinguishable effect on secondary darkening.

Table 9. Effect on digging flies of damage caused at emergence by transversecompression of abdomen with forceps {max. score = 30)

Darkening score

i"S hr. 2 hr. 3-5 hr. 6 hr. 19 hr.Abdomens compressed 3 11J 21 i 29\ 29^Controls: CO, only o i\ 4J 6 9$

(5) Ligature experiments

We are now in a position to reconsider the results of Franekel's (1935) ligatureexperiments. It has been shown (Cottrell, 1962a) that expansion and hardening anddarkening occur in the absence of 'disturbing' stimuli rather than that they areelicited by a particular stimulus situation. The placing of a ligature around a newlyemerged fly thus constitutes a very effective way of causing the inhibition of expansionbut this will not of course prevent the appearance of patchy secondary darkeningbetween 7 and 25 hr. after emergence. Fraenkel notes that ' identically treated speci-mens behaved differently' and that' even within one part, the coloration was not alwaysevenly distributed' suggesting that in some cases, at least, he was dealing withsecondary darkening and not darkening initiated by the normal physiological path.This is confirmed by the remark that ' only in flies which have been ligatured at thetime of expansion of the wings or shortly before was coloration of all parts invariablynormal'.

Evidently ligature experiments can give unequivocal results only if the followingconditions are fulfilled: (1) the fly has shown signs (by swallowing air and attemptingto expand) of initiating hardening and darkening in the normal manner; (2) darkeningis assessed not later than 3 hr. after the appearance of air-pumping; (3) the ligature isplaced so as not to deprive any part of the fly with its connexion to the spiracles;(4) the ligature is placed so as to cause a minimum of damage to any internal organ.

Imaginal ecdysis and cutiadar control of blowflies 409

Fraenkel placed his ligatures between the head and thorax, in the middle of thethorax and between the thorax and abdomen. The head contains no spiracles so thatthe first position will eliminate its air supply. Placing a ligature of requisite tightnessin the centre of the thorax will crush the thoracic ganglia, probably causing a damagereaction and possibly releasing any active substances contained in them. Neither ofthese objections holds to the same degree for a ligature placed between the thoraxand abdomen and it was decided to repeat Fraenkel's experiments using this position.In early experiments attempts were made to overcome the inhibition caused by thepresence of the ligature by means of light etherization (Fraenkel, 1935; Cottrell,1962a) but even so the initiation of air pumping occurred in only a few individuals.Later it was found that if fine steel wire ligatures were applied under CO2 anaesthesiaand the flies then suspended and given cotton-wool balls to hold, most individualswould initiate expansion.

The results obtained from a batch of twenty Caltiphora ligated at emergence andkept at 20° C. are typical and will be described here. Nine individuals extended theirwings completely, but in the remaining eleven expansion was imperfect and the elbowbends in the costal margins remained unstraightened. This imperfect expansion madethe times from recovery to extension difficult to estimate but the mean (+ s.E.) value(51-8 ± 2-i min.) as well as the mean for the times from expansion to first darkening ofthe thorax (78-0 ± 4-3 min.) closely resemble those obtained for flies deprived of theirabdomina at emergence (see Table 6). In no case was expansion of the abdomenobserved nor did darkening of the abdomen occur concurrently with that of the headand thorax. Indeed, in thirteen individuals the abdomina were still quite pale I2hr.after emergence. In the remaining seven patchy darkening appeared between 75 and620 (mean = 254) min. after the first signs had appeared in the thorax and graduallyincreased in intensity over the next few hours. In the light of the pinching experimentsdescribed above it seems reasonable to assume that this patchy darkening was adamage reaction consequent on the placing of the ligature.

Flies in which the ligatures were placed 15-20 min. after emergence darkeneduniformly and simultaneously all over their bodies, and flies in which the abdomen hadbeen denervated by means of a tight ligature tied at emergence and then removed soas to restore circulation also darkened uniformly and normally, but in this case thearea immediately under the ligature (where the epidermal cells had been crushed)failed to darken.

Taken together these results suggest that if the blood-borne darkening factor isreleased from a localized source this source is not in the abdomen. There remains thepossibility that if it is released from tissues spread throughout the body the ligaturewould effectively denervate the abdomen so preventing release in that part of the body.However, such a mechanism seems unlikely if only because it would eliminate almostall the economies in organization which are characteristic of hormonal control.

Discussion of the present results will be postponed until a subsequent paper(Cottrell, 19626).

4IO C. B. COTTRELL

SUMMARY

1. Blood transfusion experiments show that normal hardening and darkening at theimaginal ecdysis of CaUiphora erythrocephala (Meigen) is brought about by the releaseinto the blood of an active factor. Introduction of this factor into a newly emerged flysome 35 min. prior to the time at which it would normally be released is sufficient toprevent expansion.

2. The factor is normally released some 45 min. before the appearance of the firstsigns of darkening and between 3 and 15 min. after the fly has reached conditionssuitable for expansion, that is at about the time of initiation of air-pumping.

3. Decapitation at emergence will prevent the initiation of normal hardening anddarkening but not of secondary darkening. Evidently the head is concerned in therelease or the control of the release of the blood-borne darkening factor.

4. The critical period for the prevention of normal hardening and darkening bydecapitation lies between 3 and 15 min. after the fly has reached conditions suitable forexpansion.

5. Isolated abdomina behave in a manner similar to decapitated flies but theirreactions are complicated by secondary darkening associated with damage.

6. Flies deprived of their abdomina will expand at least partially but the rate oftheir hardening and darkening is reduced.

7. Damage reactions resembling secondary darkening in digging flies are moreextensive after damage to internal organs such as the gut than to superficial organs suchas the body wall.

8. Allowing for the effects of secondary darkening it is possible to demonstrate theoccurrence of the blood-borne darkening factor by means of ligatures placed atemergence between the thorax and the abdomen. Under these conditions only thehead and thorax exhibit normal darkening.

This paper represents part of a thesis submitted for the Ph.D. degree in theUniversity of Cambridge. My especial thanks are due to Prof. V. B. Wigglesworth forencouragement and supervision, and to Dr E. Bursell who read the manuscript. Thework was performed during the tenure of an 1851 Overseas Research Scholarship, andI wish to express my gratitude to the Royal Commission for the Exhibition of 1851for this generous support.

REFERENCES

COTTHELL, C. B. (1962a). General observations on the imaginal ecdysis of blowflies. Trans. R. Ent.Soc. Land, (in the Press).

COTTRELL, C. B. (19626). The imaginal ecdysis of blowflies. Detection of the blood-borne darkeningfactor and determination of some of its properties. J. Exp. Biol. (in the Press).

FRABNKKL, G. (1935). Observations and experiments on the blowfly (Calliphora erythrocephala) duringthe first day after emergence. Proc. Zool. Soc. Land. pp. 893-904.

FRAENKEL, G. (1940). Utilization and digestion of carbohydrates in the adult fly. J. Exp. Biol. 18,18-19.

VAN DER KLOOT, G. & WILLIAMS, C. M. (1954). Cocoon construction by the Cecropia silkworm.III. The alteration of spinning behaviour by chemical and surgical techniques. Behaviour, 6,233-54-

SOSSMAN, A. S. (1952). Tyrosinase and the respiration of pupae of Platysamia cecropia L. Biol. Bull.Woods Hole, 102, 30-47.

WILLIAMS, C. M. (1946). Continuous anaesthesia for insects. Science, 103, 57-9.

Journal of Experimental Biology, 39, No. 3 Plate 1

C. B. COTTRELL {Facing p. 4")

Imaginal ecdysis and cuticular control of blowflies 411

EXPLANATION OF PLATE

Fig. 1. Newly emerged flies after completion of hardening and darkening subsequent to being trans-fused with (a) active blood, (ft) 'newly emerged' blood. Note lack of expansion in (a).Fig. 2 a, b. Fly immediately on removal from digging tube after digging for 2 hr. subsequent to beingtransfused with ' newly emerged' blood. No expansion or hardening and darkening.Fig. 3 a, b. Fly similar to fig. 2 but after being allowed to complete normal expansion and hardeningand darkening.Fig. 4 a, b. Fly immediately on removal from digging tube after digging for 2 hr. subsequent to beingtransfused with active blood. Hardening and darkening completed without expansion.Fig. 5 a, 6. Fly similar to fig. 4, but after being allowed to swallow air in an attempt to expand. Notedistension of ptilinal and rostral regions, neck, membranous areas of abdomen and bladder-like rightwing filled with blood.