VOL. VI, No. 3 MARCH 1929

STUDIES IN THE METABOLISM OF INSECTMETAMORPHOSIS

BY J. G. H. FREW.

(From the Laboratories of the Research Hospital, Cambridge.)

{Received ist August 1927.)

(With Seven Text-figures.)

THE present study constitutes an attempt to correlate the processes of insectmetamorphosis with certain of the more obvious metabolic changes characteristic oflarval and pupal life. Throughout this work it has become obvious that the chemicalconstitution of different batches of larvae or pupae may be very different althoughall composed of individuals of the same age. One particular instance of this maybe cited at the outset. Two batches of larvae, A and B, were reared under almostidentical conditions, A being from slightly earlier ovipositions and reaching maturityabout two days before B. Both were reared at a somewhat low temperature untilnearly mature and the only differences between the two batches were that the highertemperature would commence nearer maturity for batch A than for batch B, andthat the sand in which A were reared was somewhat more moist than was the casefor larvae B. Both batches pupated very slowly. In each case samples of the bodyfluid were taken on the day when the majority of the larvae had evacuated the gut,and again two days later. Not only was there marked differences in the physicalproperties of the body fluids but there was also a difference in colour: the fluidof specimens A being yellow and deeply pigmented, whereas that of B had thenormal greenish tinge and was less pigmented.

Table I.

A(i)(2)

B(i)(2)

Freezingpoint

depression°C.

o-8u0-760

0719Not sufficiei

Specificgravity

i'O4351-0441

1-0415it fluid

Viscosityindex

i-85i-8o

i-54i-77

Surfacetensionindex

1-6261-3731-0621-093

Osmoticpressure

of colloidsmm. Hg.

18-020-720-824-6

pH

6-90

6-44

(1) First sample of body fluid. (2) Second sample.

The pH. of the pupae up to 24 hours old was 7-4 for pupae from both batches.The differences between the body fluid of the two batches are very striking,

particularly as regards the surface tension and the freezing point depression.J4

Ir-5329

206 J. G. H . F R E W

Similar striking differences in respect of other stages of the life history are shownin various parts of this paper. These differences increase considerably the difficultyof obtaining a "normal" set of metabolic values from which to work. Mammalianand avian predominance among vertebrates is usually partially attributed totheir ability to maintain an almost constant internal environment for their tissuesin spite of very considerable changes in external environment. It seems on thecontrary that insects—at all events the blow-fly—are distinguished by the abilityof their tissues to withstand a wide degree of variation in their environmentalconditions.

The writer has no new theory of the causation of metamorphosis to advance.The stages studied are in any case not sufficiently early to throw light on thebeginning of this process, they only indicate a few of the changes accompanyingthe process itself. The reason for the cessation of larval feeding is bound up withthe obscure problem of the cessation of growth in the higher animals in general.There are, nevertheless, a few speculations of passing interest.

When the blow-fly larva ceases to feed, its crop and gut are both markedly distended.These empty gradually as the digestible portion of their contents is absorbed and theindigestible remainder voided. The relatively hard chitinous integument allows but littledecrease in the diameter of the larva—in which it contrasts markedly with such a larvaas that of Tenebrio. When a Tenebrio larva is starved, its sternites become markedlyretracted towards the tergites to accommodate the decrease in contents of the larval body.This is possible because of the delicate nature of the pleural region uniting sternites andtergites. This differentiation of the regions of the segments is absent in the blow-fly larvaso that no considerable degree of dorso-ventral compression is possible.

After the evacuation of the gut contents the blow-fly larva contracts very markedlyin an antero-posterior direction. This may wTell be an adaptive movement tending tomaintain the internal pressure of the body fluid in spite of the diminution in volume ofthe body contents. There is no doubt that this internal pressure is about as high in anewly formed pupa as it is in an adult larva, and it diminishes during the pupal period.Attempts to measure accurately the changes in internal pressure were unsuccessful.Whether changes of internal pressure are of importance in the phenomena of metamor-phosis is not known, for no investigation of this point has yet been made.

In view of the above difficulties, particularly that involved by the high variability ofindividual organisms of the same age, it is unwise to place too much weight on experimentswhose nature precludes the use of a very large number of individuals.

i. RESPIRATION.

From a study of the respiratory level and the respiratory coefficient some lightmay be thrown on the nature or intensity of the metabolic changes associatedwith metamorphosis.

The method used for the study of the respiration was that elaborated byStephenson and Whetham (1925). The experiments were carried out and theweighings done in a small room kept at a constant temperature of 200 C.

Respiration experiments have been carried out extensively only during the

Studies in the Metabolism of Insect Metamorphosis 207

pupal period. During this period respiration is uncomplicated by muscular move-ment, feeding, and (probably) excretion. To investigate the respiration of thelarvae a very special technique is required involving the rearing of bacteria freelarvae on special media. Experiments were carried out with 100 washed, surfacesterilised (with corrosive sublimate) and dried puparia and the experiment wascarried on until the beginning of the emergence of the flies when the weighingsceased. Except in the earliest experiments the air current was continued until allflies had emerged; the <J<J and $$ were then counted.

It is significant that parallel experiments gave closely similar results. In thefollowing three experiments (for all of which the pupae were obtained from thesame batch), this is shown to be the case. In Exps. A and B the pupae were keptin the light; in Exp. C they were kept in the dark.

Table II .

ABC

O2 uptake per1 gm. of pupae

c.c.6i-862-263-8

CO2 output per1 gm. of pupae

c.c.39'84i43'2

H2O output per1 gm. of pupae

c.c.94'98I - I81-1

Mean R.Q. overpupal period

0-6440-684o-66i

The differences are probably experimental or sampling variations. The mostprobable experimental difference is the rate of the air current since this has someeffect on respiration. These differences are relatively small and in no way accountfor the differences mentioned below. The above experiment demonstrates also thatlight has no appreciable effect on the metabolism of pupal respiration.

In Figs. 1-4 are given the details (in graphical form) of three respirationexperiments. These experiments are not those quoted in Table II.

The graphs (Figs. 1-4) show that while the puparia in Exps. A and C wereclosely similar as regards respiration, the puparia in Exp. B had a markedly lowerrespiratory quotient, lower O2 intake and CO2 output and a very much higherH2O output. This last is not an invariable accompaniment of low R.Q.'S as low O2

intake and CO2 output appear to be. The interesting point about these experimentsis that whereas A and C contained a marked predominance of g puparia, thepuparia in B were predominantly $.

Exp. A may be taken as typical for pupal respiration. The only point to whichattention need particularly be drawn is the low R.Q.; the mean R.Q. over the wholepupal period is 0-651. It is possible that the low respiratory quotient is due to anincomplete oxidation of the fats. Acetone bodies may be instanced as such in-complete oxidation products which can actually be detected in aqueous extracts ofthe puparia. On the other hand, fat estimations indicate that fat is not used inrespiration to any great extent during the first half of the pupal period. Theformation of carbohydrate from either fat or protein would result in a low R.Q.There is definite evidence (see below) of a vigorous synthesis of glucose during the

14-2

208 J. G. H . F R E W

whole of the pupal period, and at present this must be regarded as the mostprobable explanation of the low R.Q. values obtained.

Table III.

Weight ofioo larvae

gm.8-92049-36128*93749=1265

Per 1 gm. of larvae

O2 intake

c.c.13-5

4'97*37.9

CO2 output

c.c.io-o

3'S5*55-8

H2O output

c.c.7-6

II-O2-53-0

R.Q.

0-741O-7O2O-7530739

Pupation

/o37272919

0-9-

1——O—' 2 ^2 3J. _ x-L2 4 2

2 "25^-62 72

Age of Pupae in DaysFig. 1.

2 • *-» g 112 • *•©9"2~10"2 11-J—12-J

2. DISTRIBUTION OF FREE CARBOHYDRATES.

In order to determine what part, if any, carbohydrates play in the respiratorycycle, an attempt has been made to trace the distribution of reducing sugars, and offree glycogen (during the process of pupation) firstly in the body fluids, andsecondly in whole organism.

(a) Carbohydrates in the body fluid.

The reducing power of the body fluid of the various stages of larvae and pupaeis expressed as milligrams glucose per 100 c.c. of body fluid. The method used forthe estimation was that of Hagedorn and Jensen. That reducing substances other

Studies in the Metabolism of Insect Metamorphosis 209

than glucose may have been present and affected the results is possible. Glucoseitself is undoubtedly present; the osazone has been obtained and all fructose testshave proved negative. The titration method was not checked polarimetrically,

Fig. 2.

11

§10

e 8o -i

IS 5

so

a 3

I 2ou

1

/ •

f tEM for £Mfor

1-1| | 2J-8J |4i-5j I 6i-7i • -5-"«AandB C

Age of pupae in DaysFig. 3.

largely owing to the considerable difficulties experienced in obtaining a clear andcolourless solution of the reducing substance in sufficient concentration to allowof a trustworthy polarimetric reading. All glucose determinations were done inquadruplicate.

210 J. G. H . F R E W

The greater part of larval growth in Cyclorrhapha occurs during the thirdlarval stadium. When fully grown—or under certain conditions before being fullygrown—the larvae cease feeding and bury themselves in the soil. At this time thecrop and gut are both greatly distended with undigested food. Between the time

21

20

19

18

17

^ 165-1

1 15^ 14

o 1303a3 19

2 11

- B. •

0* 9 -

o 7 -

K 6 -

4 -

3 -

2 -

1 -

it

ii

i

t

ft1 T1

62~72

EM for EM forAandB C ,

5 I 3-2-4^ 5^-6^ 7|-8^ 9 -̂10^ 111 -12-̂ 13-̂ -14̂ 15 -̂16^

Age of Pupae in Days

Fig. 4.

when the larvae finish feeding and the time when they pupate a variable timeelapses and before pupation the crop and gut are completely emptied of theircontents—this usually happening one or two days before pupation. For any largebatch of larvae (though all may have commenced development simultaneously) alldo not pupate upon the same day.

Studies in the Metabolism of Insect Metamorphosis 211

In Fig. 5, showing the "glucose" value of the larval body fluid, F marks theday upon which the larvae finished feeding, G the day upon which the gut becamecompletely empty and P marks the first day upon which the first larvae pupated.The values shown on any one graph are those obtained on successive days fromthe same batch of larvae.

Examining first the two "normal" curves (B and E) it will be seen that towardsthe end of the feeding period the glucose value of the blood falls rapidly. It isimpossible to obtain values for the very youngest larvae owing to the impossibilityof obtaining sufficient body fluid with certainty of not having punctured the gut.

210

200

| 190

•I4 180

§ 1 7°o2 isof-

2 1 5 ( *8•§. 140to

130

120

110

Graph E

Graph A

G

1 8 10 11 12 13 142 3 4 5 6 7Days

F. Finished feeding. G. Gut empty. P. Beginning of pupation.Fig. 5-

At the end of the feeding period the fall in glucose value continues for a variablenumber of days. Before the gut is emptied the glucose value rises sharply andcontinues to rise to a maximum on the day on which pupation begins, afterwhich it again falls sharply.

Comparing now the three abnormal curves, A, C and D, in which only a fewlarvae pupated, the most striking difference is that they do not exhibit the well-marked "peak" glucose value on the day of commencement of pupation—all infact show slightly higher values some days after the beginning of pupation, thoughthey are all very low. Curve A, the only complete abnormal curve, shows also norise in glucose value before the evacuation of the gut; in this respect it differs fromthe normal curves.

2i2 J . G. H . F R E W

The glucose in the body fluid of the feeding larva may reasonably be associatedwith direct absorption from the alimentary tract. As the glucose value commencesto fall before feeding is finished and as it continues to fall after the end of feedingbut before the evacuation of the gut contents, the rise in glucose value which occursshortly prior to this evacuation cannot be ascribed to absorption from the gut; andthe rise in glucose value after the evacuation of the gut contents can obviously havenothing to do with absorption from the gut.

Of the fall in glucose value towards the end of the feeding period and immediatelyafterwards no well-grounded explanation can be advanced at present as no re-spiration experiments have been carried out on the early larval stages.

Histolysis of the larval tissues undoubtedly commences some time prior topupation and the commencement of the rise in the glucose value of the body fluidmay possibly be one of the first manifestations of the beginning of this process.It seems a little significant that three batches of larvae which failed to show a risein the glucose value of the body fluid at the period when pupation was commencingfailed also to pupate with normal rapidity when pupation did commence. It isprobable that changes in glucose concentration have some connection with pupation,though the connection may not be one of cause and effect. Above, three "peak"glucose values for larval body fluid are shown by an X (Fig. 5), the glucose valuesof the body fluid of "white" pupae, i.e. very young pupae representing a stage ofdevelopment only an hour or two later than that of the larvae among which theywere found. It will be seen that these values are slightly higher than the peaklarval values but very considerably lower than the values for pupae up to 24 hoursold (Fig. 6).

In Fig. 6 are shown the changes occurring in the glucose content of the bodyfluid during the pupal period. The points lying on one curve were obtained fromthe body fluid of pupae derived from the same batch of larvae—though notnecessarily from pupae pupating on the same day. In one case two values are givenfor pupae of the same age; the lower value is for the first pupae, and the highervalue for the pupae of the main pupal day—from which were also obtained theother values on this particular curve. A few isolated values are also shown.

The most noteworthy fact about the glucose value of the pupal body fluid isthat it is between three and four times the glucose value of the larval body fluid.This enormous rise in glucose value occurs during the first 24 hours of pupal lifeand may be regarded as a continuation and acceleration of the process by whichthe glucose value of the larval body fluid rises rapidly during the last two or threedays of larval life.

The origin of the glucose by which this change is effected is not known withany certainty. It is probably derived from the histolysis of the larval tissues. Itwill be noted that there are two peak values for the glucose content of the pupalbody fluid; one at 3 to 4 days old, and the other (on the complete curve) at 7 to8 days old. These two peaks are believed to be connected with a synthesis of glucosefrom fat or protein. The second peak occurs immediately after the day of maximumosmotic pressure of the colloids which has provisionally been regarded as evidence

Studies in the Metabolism of Insect Metamorphosis 213

of protein breakdown. During the last three days of pupal life the glucose valueof the pupal body fluid falls very rapidly.

It should be mentioned here that there is no glycogen in the larva or pupa, andno storage of any carbohydrate which, by acid hydrolysis, can be converted intoglucose.

0-1 8-9 10-117-8 9-10 11-12

2-3 4-5 6-71 -2 3-4 5-6

Age of Pupae in Days

Fig. 6.

(b) Carbohydrates in entire larva and pupa.

Parallel with the above experiments are those which show the amount of freecarbohydrate in the intact larva or pupa.

Owing to the small number of pupae used for the extractions, the successivedeterminations through the whole pupal period can be made with pupae whichpupated on the same day. In Fig. 7 are shown the data for three batches of pupaeand their antecedent larvae. The experiments illustrated by the two lower curveswere performed at room temperature and it is to the upper one that attention ismore particularly directed. The pupae used were from the main pupation day.In the case of larvae the gut contents was evacuated before the estimations wereperformed.

The broken line shows the glucose value of the larvae for the three days priorto pupation, and for those individuals which continued as larvae after the mainpupation day.

214 J. G. H. FREW

After the evacuation of the gut contents the glucose content of the larvae risesto a maximum. Immediately upon pupation the glucose content commences tofall and during the first 24 hours of pupal life the glucose content is reduced byapproximately one-third of its value immediately prior to pupation. The values forvery young pupae (X and Y, Fig. 7) demonstrate beyond doubt that this is theactual course of events. It would otherwise be a conceivable though improbableinterpretation of the figures to say that the glucose value fell immediately prior to

700-

0-1 2-3 4-5 6-7 8-9 10-11 12-13 14-151-2 3-4 5-6 7-8 9-10 11-12 13-14

Days prior topupation (larvae)

Pupae: Firm line. Larvae: Broken line.Age of Pupae in Days

Fig. 7.

X and Y: Pupae 4-6 hrs old.

pupation. The most noteworthy features during the pupal period are the "peak"values of glucose content for pupae of 3-4 and 7-8 days. These correspond preciselywith the peak values for the glucose content of the body fluid (Fig. 6).

After the main pupal day there are many larvae left which continue to pupateon successive days. For two days the glucose content of these larvae is approxi-mately at the typical "peak" value for larvae immediately prior to pupation, andduring these two days pupation is occurring rapidly. After this time, pupationslows down and the residual larvae are probably slightly abnormal in theirmetabolism; they pupate very slowly and usually some fail to pupate at all. The

Studies in the Metabolism of Insect Metamorphosis 215

glucose value of such larvae falls to a value only slightly higher than it would havebeen if they had become pupae on the main pupal day.

It is the writer's opinion that the larval values in question do actually representa falling off in the glucose content of these larvae from a previously acquired peakvalue, and that they are not due to the presence in the population of certain larvaewhose glucose content never rises to the peak value.

The two curves P-Q and N-O (Fig. 7) show the variation in glucose content forlarvae and pupae reared at 180 C. instead of at 210 C. In both cases a good percentageof emergence was obtained, though the pupal period is naturally prolonged by thelower temperature. Comparing these two curves with the "normal" curves thefollowing points are noteworthy. Pupation can occur with a glucose content con-siderably below the normal; a fall from the pre-pupation "peak" glucose valuemay occur in the larvae before any pupation has occurred; successful emergencemay take place without the two minor peak values of glucose content shown by thenormal pupae at about the fourth and eighth days respectively of the pupal period.

There is no doubt that the amount of glucose in a pupa at the commencementof the pupal period is quite insignificant compared with the respiration which goeson during this period. 0*7 mg. of glucose would on combustion give a little over1 mg. of CO2. This is approximately the quantity of CO2 given off by one pupa duringthe first 24 hours of pupal life. Therefore either the glucose present in the pupa isnot used for the purposes of respiration, or it is replenished as rapidly as it is used.The latter is the more probable hypothesis. The three peak values of the normalcurve (Fig. 7) indicate three periods when there is an active glucose formingmetabolism; the fact that the values fall off after each "peak" shows that theglucose is utilised in some way.

As already stated there is no storage of glycogen in the larva nor is there presentin any quantity any storage carbohydrate which is convertible by acid hydrolysisinto glucose or other reducing sugar. Thus in one estimation the glucose contentof larvae estimated in the ordinary way was 0*395 mg. glucose per larva. Anextract made from crushed larvae subjected to acid hydrolysis gave a value of0*440 mg. of glucose. The difference in values is almost insignificant; it is indeedonly just outside the margin of experimental and sampling error which has beenestimated at ± 0*020 mg. glucose. Glucose is therefore not derived from a carbo-hydrate storage.

Fig. 7 shows that there are three periods of "glucose" formation, but betweenthese periods glucose might not be synthesised. If this were so, Fig. 7 woulddemonstrate that glucose was formed in only very small amounts and was probablyof little significance as regards the respiratory metabolism. An attempt wastherefore made to estimate the magnitude of glucose synthesis from day to day.The glucose value of one extract so obtained was estimated immediately as describedabove. The second extract was incubated for 8 hours at about 180 C. with constantmechanical stirring. At the end of 8 hours its glucose value was obtained. Theglucose value of the unincubated extract being taken as unity, the figures inTable III give the glucose value of the incubated extract. The method is obviously

216 J. G. H . F R E W

very imperfect. It is a big assumption to consider the intensity of and variationsin the synthesis of glucose as shown by this method to be in any way approximateto the conditions obtaining in the living pupa; nor can it be asserted that a possiblerespiratory utilisation of the glucose is entirely eliminated by this method.

With these reservations it is permissible to affirm that from the time when thelarval gut is evacuated until the time of emergence of the flies there is a constantlyactive synthesis of glucose. There is indeed sufficient glucose formed every 24 hoursto provide fuel for the pupal respiration during that period. This may indicatethat whatever the ultimate source of the respiratory fuel (fat or protein) glucoseis an intermediate stage in its oxidation, but the glucose formed may also of coursehave a function quite apart from that of respiration in the process of building upthe imaginal tissues. It is perhaps necessary to reiterate here that the method usedin the glucose estimations is not specific for glucose alone. It is a little unfortunatethat the pupae used for the two series of estimations given in Table III were keptat 180 C. instead of 210 C. as the pupal period is thereby prolonged and the pupalpeak values of glucose content depressed.

Synthesis of glucose is not necessarily accompanied by a rise in the glucosecontent of the pupae, showing that there is also an active utilisation of the glucoseformed. The fact that the more active synthesis of glucose may occur at periodswhen the amount of respiration is low, indicates that the glucose formed is atleast not entirely used as a respiratory fuel and is perhaps mainly used in ana-bolic processes.

3. FAT AND NITROGEN CONTENT OF LARVAE AND PUPAE.

Fat estimations were made by the Soxhlet method with alcohol and ether ex-traction ; the tissues were dried in an atmosphere of nitrogen. The results showed,without doubt, that during the first four or five days of pupal life there is no utilisa-tion for respiration of any of the alcohol-ether soluble constituents by the bodytissues. The earliest values available for the fat content of pupae are the valuesfor white pupae, i.e. for pupae only a few hours old. In each case the values soobtained are very markedly lower than the value of larvae obtained on the sameday. The significance of this is doubtful since in every case the newly formedpupae are considerably lighter than an equal number of larvae of the same day.It is at least doubtful whether in any one larva there is a diminution in the amountof alcohol-ether soluble substance in the tissues immediately prior to pupation.Towards the end of pupal life the fat content diminished notably and it seemslegitimate to suppose that this diminution is at least partially due to respiratoryactivities. The alcohol-ether soluble content of the larvae diminishes graduallythough its ratio to body weight increases slightly—from 7-8 to 8*3 per cent. Duringthe pupal period the ratio of fat to body weight falls very markedly: A 7-5 to 57,B 7-1 to 5-1, C 6-8 to 4-2 per cent. Estimations were made of the fat content of50 tftf and of 50 $? from B pupae. In each case alcohol-ether soluble substancemake up 5 per cent, of the body weight; the $? are slightly heavier than the

Studies in the Metabolism of Insect Metamorphosis 217

There is no adequate evidence that the respiration of <ftj and $$ is essentiallydifferent.

With regard to the possibility of a synthesis of glucose from fat it may be saidwith fair certainty that this does not occur at the end of larval life or at the beginningof pupal life. The possibility still remains that it may occur during the latter partof the pupal period.

In the absence of any excretory mechanism estimations of the total N2 contentduring the pupal period give little information, and estimations of any changes inthe amount of various possible breakdown products of protein combustion havenot been made. All that can be said is that there is no actual loss of N2 between thetime when the pupae form and the time when the flies emerge. It remains a possi-bility that nitrogenous waste products accumulate in the meconium which is passedsome hours after pupation. The N2 content of the meconium is somewhat difficultto estimate with accuracy, but it appears to be very low and almost negligible incomparison with the total N2 content of the pupae. The indications are that thereis no excretion of N2 into the meconium at all commensurate in amount with themagnitude of the pupal respiration. Therefore, if there is a protein respiratorymetabolism at any time during the pupal period, it is of such a nature that thenitrogenous end-products are retained within the tissues of the pupa.

SUMMARY AND CONCLUSIONS.

1. The low values of the Respiratory Quotient (0*65) during the pupal periodare most readily explained on the theory that some constituent of the tissues isonly partially oxidised and remains, in part at least, as a permanent constituent ofthe body instead of being oxidised to products such as CO2 and H2O which areeliminated.

2. There is definite evidence of a very marked synthesis of glucose duringpupation. If this glucose were all oxidised to CO2 and H2O, the expected RespiratoryQuotient would be about 0-7 or o-8 depending on whether the glucose is formedfrom protein or fat. Actually the Respiratory Quotient seldom rises as high as 0*7and is frequently below o*6. In certain instances it may be much lower than this.It is improbable then that the glucose formed is entirely used in respiration; somemust almost certainly be used in building up the growing imaginal tissue substance.

3. The synthetic processes which form glucose are active throughout the wholepupal period, though not uniformly so. The alcohol-ether soluble constituents ofthe body do not diminish in quantity during the first part of the pupal, period.During this period therefore the glucose formed cannot be derived from fat. Thereis no storage (in the body of mature larvae) of glycogen or any other higher carbo-hydrate convertible by acid hydrolysis into glucose (or other reducing sugar).Therefore, during the earlier part of the pupal period the glucose must be derivedfrom a protein source. The protein is used in such a way that no nitrogen is lostby the body.

218 J. G. H . F R E W

4. During the latter half of the pupal period the fat content of the bodycontinually diminishes and during this period there may be a fat to carbohydratemetabolism. The shape of the respiration curves shows a gradual diminution ofrespiration during the early part of the pupal period and a gradual increase duringthe latter half.

5. There is no reliable evidence which indicates that the metabolic processesof the two sexes are different from each other.

REFERENCES.STEPHENSON, M. AND WHETHAM, M. D. (1925). Proc. Roy. Soc. B, 95, 200.