VOL.
VI, No. 3
MARCH 1929
STUDIES IN THE METABOLISM OF INSECT
METAMORPHOSIS
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 insect
metamorphosis with certain of the more obvious metabolic changes characteristic of
larval and pupal life. Throughout this work it has become obvious that the chemical
constitution of different batches of larvae or pupae may be very different although
all composed of individuals of the same age. One particular instance of this may
be cited at the outset. Two batches of larvae, A and B, were reared under almost
identical conditions, A being from slightly earlier ovipositions and reaching maturity
about two days before B. Both were reared at a somewhat low temperature until
nearly mature and the only differences between the two batches were that the higher
temperature would commence nearer maturity for batch A than for batch B, and
that the sand in which A were reared was somewhat more moist than was the case
for larvae B. Both batches pupated very slowly. In each case samples of the body
fluid 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 physical
properties of the body fluids but there was also a difference in colour: the fluid
of specimens A being yellow and deeply pigmented, whereas that of B had the
normal greenish tinge and was less pigmented.
Table I.
Freezing
point
depression
°C.
A(i)
(2)
B(i)
(2)
Specific
gravity
o-8u
i'O435
1-0441
0-760
0719
1-0415
Not sufficieiit fluid
(1) First sample of body
Viscosity
index
Surface
tension
index
Osmotic
pressure
of colloids
mm. Hg.
i-85
i-8o
1-626
1-373
1-062
1-093
18-0
20-7
20-8
24-6
i-54
i-77
fluid.
pH
6-90
6-44
(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.
J
4
Ir-5329
206
J.
G.
H.
FREW
Similar striking differences in respect of other stages of the life history are shown
in various parts of this paper. These differences increase considerably the difficulty
of obtaining a "normal" set of metabolic values from which to work. Mammalian
and avian predominance among vertebrates is usually partially attributed to
their ability to maintain an almost constant internal environment for their tissues
in spite of very considerable changes in external environment. It seems on the
contrary that insects—at all events the blow-fly—are distinguished by the ability
of their tissues to withstand a wide degree of variation in their environmental
conditions.
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 the
beginning of this process, they only indicate a few of the changes accompanying
the process itself. The reason for the cessation of larval feeding is bound up with
the 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 the
indigestible remainder voided. The relatively hard chitinous integument allows but little
decrease in the diameter of the larva—in which it contrasts markedly with such a larva
as that of Tenebrio. When a Tenebrio larva is starved, its sternites become markedly
retracted 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 and
tergites. This differentiation of the regions of the segments is absent in the blow-fly larva
so that no considerable degree of dorso-ventral compression is possible.
After the evacuation of the gut contents the blow-fly larva contracts very markedly
in an antero-posterior direction. This may wTell be an adaptive movement tending to
maintain the internal pressure of the body fluid in spite of the diminution in volume of
the body contents. There is no doubt that this internal pressure is about as high in a
newly 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 metamorphosis 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 of
individual organisms of the same age, it is unwise to place too much weight on experiments
whose 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 light
may be thrown on the nature or intensity of the metabolic changes associated
with metamorphosis.
The method used for the study of the respiration was that elaborated by
Stephenson and Whetham (1925). The experiments were carried out and the
weighings 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 movement, feeding, and (probably) excretion. To investigate the respiration of the
larvae a very special technique is required involving the rearing of bacteria free
larvae on special media. Experiments were carried out with 100 washed, surface
sterilised (with corrosive sublimate) and dried puparia and the experiment was
carried on until the beginning of the emergence of the flies when the weighings
ceased. Except in the earliest experiments the air current was continued until all
flies had emerged; the <J<J and $$ were then counted.
It is significant that parallel experiments gave closely similar results. In the
following three experiments (for all of which the pupae were obtained from the
same batch), this is shown to be the case. In Exps. A and B the pupae were kept
in the light; in Exp. C they were kept in the dark.
Table II.
A
B
C
O2 uptake per
1 gm. of pupae
CO2 output per
1 gm. of pupae
H2O output per
1 gm. of pupae
c.c.
6i-8
62-2
63-8
c.c.
39'8
c.c.
94'9
4i
43'2
8I-I
81-1
Mean R.Q. over
pupal period
0-644
0-684
o-66i
The differences are probably experimental or sampling variations. The most
probable experimental difference is the rate of the air current since this has some
effect on respiration. These differences are relatively small and in no way account
for the differences mentioned below. The above experiment demonstrates also that
light has no appreciable effect on the metabolism of pupal respiration.
In Figs. 1-4 are given the details (in graphical form) of three respiration
experiments. These experiments are not those quoted in Table II.
The graphs (Figs. 1-4) show that while the puparia in Exps. A and C were
closely similar as regards respiration, the puparia in Exp. B had a markedly lower
respiratory quotient, lower O2 intake and CO2 output and a very much higher
H2O 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 experiments
is that whereas A and C contained a marked predominance of g puparia, the
puparia in B were predominantly $.
Exp. A may be taken as typical for pupal respiration. The only point to which
attention need particularly be drawn is the low R.Q.; the mean R.Q. over the whole
pupal period is 0-651. It is possible that the low respiratory quotient is due to an
incomplete oxidation of the fats. Acetone bodies may be instanced as such incomplete oxidation products which can actually be detected in aqueous extracts of
the puparia. On the other hand, fat estimations indicate that fat is not used in
respiration to any great extent during the first half of the pupal period. The
formation 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.
FREW
whole of the pupal period, and at present this must be regarded as the most
probable explanation of the low R.Q. values obtained.
Table III.
Per 1 gm. of larvae
Weight of
ioo larvae
Pupation
R.Q.
gm.
8-9204
9-3612
8*9374
9=1265
O 2 intake
CO 2 output
H 2 O output
c.c.
13-5
4'9
7*3
7.9
c.c.
io-o
3'S
5*5
5-8
c.c.
7-6
0-741
O-7O2
O-753
0739
II-O
2-5
3-0
/o
37
27
29
19
0-9-
1——O—
'2
^2
3 J.2 _ 4x-L
2
2 "2
5^-62
72
2
9
• *-» g
"2~ 1 0 "2
112
• *•©
11-J—12-J
Age of Pupae in Days
Fig. 1.
2. DISTRIBUTION OF FREE CARBOHYDRATES.
In order to determine what part, if any, carbohydrates play in the respiratory
cycle, an attempt has been made to trace the distribution of reducing sugars, and of
free glycogen (during the process of pupation) firstly in the body fluids, and
secondly in whole organism.
(a) Carbohydrates in the body fluid.
The reducing power of the body fluid of the various stages of larvae and pupae
is expressed as milligrams glucose per 100 c.c. of body fluid. The method used for
the 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. Glucose
itself is undoubtedly present; the osazone has been obtained and all fructose tests
have proved negative. The titration method was not checked polarimetrically,
Fig. 2.
11
§10
e 8
o
-i
S
5
I
/
so
•
a 3
I 2
o 1
u
f
t
EM for £Mfor
AandB C
1-1| | 2J-8J |4i-5j I 6i-7i • -5-"«
Age of pupae in Days
Fig. 3.
largely owing to the considerable difficulties experienced in obtaining a clear and
colourless solution of the reducing substance in sufficient concentration to allow
of a trustworthy polarimetric reading. All glucose determinations were done in
quadruplicate.
210
J. G. H .
FREW
The greater part of larval growth in Cyclorrhapha occurs during the third
larval stadium. When fully grown—or under certain conditions before being fully
grown—the larvae cease feeding and bury themselves in the soil. At this time the
crop and gut are both greatly distended with undigested food. Between the time
21
20 - B.
•
19
18
17
^ 16
i
t
5-1
1 15
i
i
i
^ 14
o 13
03
a
3
19
t
211
0*
9-
o
7-
K
6-
4321-
EM for EM for
AandB C ,
ft1
6
T1
2~ 7 2
5 I
3-2-4^
5^-6^
7|-8^
9^-10^ 111 -12-^ 13-^-14^ 15^-16^
Age of Pupae in Days
Fig. 4.
when the larvae finish feeding and the time when they pupate a variable time
elapses and before pupation the crop and gut are completely emptied of their
contents—this usually happening one or two days before pupation. For any large
batch of larvae (though all may have commenced development simultaneously) all
do 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 the
day upon which the larvae finished feeding, G the day upon which the gut became
completely 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 from
the same batch of larvae.
Examining first the two "normal" curves (B and E) it will be seen that towards
the end of the feeding period the glucose value of the blood falls rapidly. It is
impossible to obtain values for the very youngest larvae owing to the impossibility
of obtaining sufficient body fluid with certainty of not having punctured the gut.
Graph E
210
200
|
190
•I 4 180
Graph A
§17°
o
2 isof2
8
15(
*
•§. 140
to
130
120
110
G
1
2
F. Finished feeding.
3
4
5
6
7
Days
G. Gut empty.
Fig. 5-
8
10
11
12
13
14
P. Beginning of pupation.
At the end of the feeding period the fall in glucose value continues for a variable
number of days. Before the gut is emptied the glucose value rises sharply and
continues to rise to a maximum on the day on which pupation begins, after
which it again falls sharply.
Comparing now the three abnormal curves, A, C and D, in which only a few
larvae pupated, the most striking difference is that they do not exhibit the wellmarked "peak" glucose value on the day of commencement of pupation—all in
fact show slightly higher values some days after the beginning of pupation, though
they are all very low. Curve A, the only complete abnormal curve, shows also no
rise in glucose value before the evacuation of the gut; in this respect it differs from
the normal curves.
2i2
J.
G.
H.
FREW
The glucose in the body fluid of the feeding larva may reasonably be associated
with direct absorption from the alimentary tract. As the glucose value commences
to fall before feeding is finished and as it continues to fall after the end of feeding
but before the evacuation of the gut contents, the rise in glucose value which occurs
shortly prior to this evacuation cannot be ascribed to absorption from the gut; and
the rise in glucose value after the evacuation of the gut contents can obviously have
nothing to do with absorption from the gut.
Of the fall in glucose value towards the end of the feeding period and immediately
afterwards no well-grounded explanation can be advanced at present as no respiration experiments have been carried out on the early larval stages.
Histolysis of the larval tissues undoubtedly commences some time prior to
pupation and the commencement of the rise in the glucose value of the body fluid
may 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 rise
in the glucose value of the body fluid at the period when pupation was commencing
failed also to pupate with normal rapidity when pupation did commence. It is
probable 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 values
of the body fluid of "white" pupae, i.e. very young pupae representing a stage of
development only an hour or two later than that of the larvae among which they
were found. It will be seen that these values are slightly higher than the peak
larval values but very considerably lower than the values for pupae up to 24 hours
old (Fig. 6).
In Fig. 6 are shown the changes occurring in the glucose content of the body
fluid during the pupal period. The points lying on one curve were obtained from
the body fluid of pupae derived from the same batch of larvae—though not
necessarily from pupae pupating on the same day. In one case two values are given
for pupae of the same age; the lower value is for the first pupae, and the higher
value for the pupae of the main pupal day—from which were also obtained the
other 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 is
that 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 life
and may be regarded as a continuation and acceleration of the process by which
the glucose value of the larval body fluid rises rapidly during the last two or three
days of larval life.
The origin of the glucose by which this change is effected is not known with
any certainty. It is probably derived from the histolysis of the larval tissues. It
will be noted that there are two peak values for the glucose content of the pupal
body fluid; one at 3 to 4 days old, and the other (on the complete curve) at 7 to
8 days old. These two peaks are believed to be connected with a synthesis of glucose
from fat or protein. The second peak occurs immediately after the day of maximum
osmotic 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 value
of the pupal body fluid falls very rapidly.
It should be mentioned here that there is no glycogen in the larva or pupa, and
no storage of any carbohydrate which, by acid hydrolysis, can be converted into
glucose.
0-1
2-3
1 -2
4-5
6-7
3-4
5-6
Age of Pupae in Days
8-9
7-8
10-11
9-10
11-12
Fig. 6.
(b) Carbohydrates in entire larva and pupa.
Parallel with the above experiments are those which show the amount of free
carbohydrate in the intact larva or pupa.
Owing to the small number of pupae used for the extractions, the successive
determinations through the whole pupal period can be made with pupae which
pupated on the same day. In Fig. 7 are shown the data for three batches of pupae
and their antecedent larvae. The experiments illustrated by the two lower curves
were performed at room temperature and it is to the upper one that attention is
more particularly directed. The pupae used were from the main pupation day.
In the case of larvae the gut contents was evacuated before the estimations were
performed.
The broken line shows the glucose value of the larvae for the three days prior
to pupation, and for those individuals which continued as larvae after the main
pupation day.
214
J. G. H. FREW
After the evacuation of the gut contents the glucose content of the larvae rises
to a maximum. Immediately upon pupation the glucose content commences to
fall and during the first 24 hours of pupal life the glucose content is reduced by
approximately one-third of its value immediately prior to pupation. The values for
very young pupae (X and Y, Fig. 7) demonstrate beyond doubt that this is the
actual course of events. It would otherwise be a conceivable though improbable
interpretation of the figures to say that the glucose value fell immediately prior to
700-
0-1
Days prior to
pupation (larvae)
1-2
2-3
3-4
4-5
5-6
6-7
7-8
8-9
10-11 12-13
14-15
9-10
11-12
13-14
Pupae: Firm line. Larvae: Broken line. X and Y: Pupae 4-6 hrs old.
Age of Pupae in Days
Fig. 7.
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 precisely
with 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 pupate
on successive days. For two days the glucose content of these larvae is approximately at the typical "peak" value for larvae immediately prior to pupation, and
during these two days pupation is occurring rapidly. After this time, pupation
slows down and the residual larvae are probably slightly abnormal in their
metabolism; 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 have
been if they had become pupae on the main pupal day.
It is the writer's opinion that the larval values in question do actually represent
a falling off in the glucose content of these larvae from a previously acquired peak
value, and that they are not due to the presence in the population of certain larvae
whose glucose content never rises to the peak value.
The two curves P-Q and N-O (Fig. 7) show the variation in glucose content for
larvae and pupae reared at 180 C. instead of at 21 0 C. In both cases a good percentage
of emergence was obtained, though the pupal period is naturally prolonged by the
lower temperature. Comparing these two curves with the "normal" curves the
following points are noteworthy. Pupation can occur with a glucose content considerably below the normal; a fall from the pre-pupation "peak" glucose value
may occur in the larvae before any pupation has occurred; successful emergence
may take place without the two minor peak values of glucose content shown by the
normal 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 commencement
of the pupal period is quite insignificant compared with the respiration which goes
on during this period. 0*7 mg. of glucose would on combustion give a little over
1 mg. of CO 2 . This is approximately the quantity of CO2 given off by one pupa during
the first 24 hours of pupal life. Therefore either the glucose present in the pupa is
not 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 normal
curve (Fig. 7) indicate three periods when there is an active glucose forming
metabolism; the fact that the values fall off after each "peak" shows that the
glucose is utilised in some way.
As already stated there is no storage of glycogen in the larva nor is there present
in any quantity any storage carbohydrate which is convertible by acid hydrolysis
into glucose or other reducing sugar. Thus in one estimation the glucose content
of larvae estimated in the ordinary way was 0*395 mg. glucose per larva. An
extract made from crushed larvae subjected to acid hydrolysis gave a value of
0*440 mg. of glucose. The difference in values is almost insignificant; it is indeed
only just outside the margin of experimental and sampling error which has been
estimated at ± 0*020 mg. glucose. Glucose is therefore not derived from a carbohydrate storage.
Fig. 7 shows that there are three periods of "glucose" formation, but between
these periods glucose might not be synthesised. If this were so, Fig. 7 would
demonstrate that glucose was formed in only very small amounts and was probably
of little significance as regards the respiratory metabolism. An attempt was
therefore made to estimate the magnitude of glucose synthesis from day to day.
The glucose value of one extract so obtained was estimated immediately as described
above. The second extract was incubated for 8 hours at about 180 C. with constant
mechanical stirring. At the end of 8 hours its glucose value was obtained. The
glucose value of the unincubated extract being taken as unity, the figures in
Table III give the glucose value of the incubated extract. The method is obviously
216
J.
G.
H.
FREW
very imperfect. It is a big assumption to consider the intensity of and variations
in the synthesis of glucose as shown by this method to be in any way approximate
to the conditions obtaining in the living pupa; nor can it be asserted that a possible
respiratory utilisation of the glucose is entirely eliminated by this method.
With these reservations it is permissible to affirm that from the time when the
larval gut is evacuated until the time of emergence of the flies there is a constantly
active synthesis of glucose. There is indeed sufficient glucose formed every 24 hours
to provide fuel for the pupal respiration during that period. This may indicate
that whatever the ultimate source of the respiratory fuel (fat or protein) glucose
is an intermediate stage in its oxidation, but the glucose formed may also of course
have a function quite apart from that of respiration in the process of building up
the imaginal tissues. It is perhaps necessary to reiterate here that the method used
in the glucose estimations is not specific for glucose alone. It is a little unfortunate
that the pupae used for the two series of estimations given in Table III were kept
at 180 C. instead of 21 0 C. as the pupal period is thereby prolonged and the pupal
peak values of glucose content depressed.
Synthesis of glucose is not necessarily accompanied by a rise in the glucose
content of the pupae, showing that there is also an active utilisation of the glucose
formed. The fact that the more active synthesis of glucose may occur at periods
when the amount of respiration is low, indicates that the glucose formed is at
least not entirely used as a respiratory fuel and is perhaps mainly used in anabolic processes.
3. FAT AND NITROGEN CONTENT OF LARVAE AND PUPAE.
Fat estimations were made by the Soxhlet method with alcohol and ether extraction ; 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 utilisation for respiration of any of the alcohol-ether soluble constituents by the body
tissues. The earliest values available for the fat content of pupae are the values
for white pupae, i.e. for pupae only a few hours old. In each case the values so
obtained are very markedly lower than the value of larvae obtained on the same
day. The significance of this is doubtful since in every case the newly formed
pupae 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 amount
of alcohol-ether soluble substance in the tissues immediately prior to pupation.
Towards the end of pupal life the fat content diminished notably and it seems
legitimate to suppose that this diminution is at least partially due to respiratory
activities. The alcohol-ether soluble content of the larvae diminishes gradually
though its ratio to body weight increases slightly—from 7-8 to 8*3 per cent. During
the pupal period the ratio of fat to body weight falls very markedly: A 7-5 to 5 7 ,
B 7-1 to 5-1, C 6-8 to 4-2 per cent. Estimations were made of the fat content of
50 tftf and of 50 $? from B pupae. In each case alcohol-ether soluble substance
make 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 essentially
different.
With regard to the possibility of a synthesis of glucose from fat it may be said
with fair certainty that this does not occur at the end of larval life or at the beginning
of pupal life. The possibility still remains that it may occur during the latter part
of the pupal period.
In the absence of any excretory mechanism estimations of the total N2 content
during the pupal period give little information, and estimations of any changes in
the amount of various possible breakdown products of protein combustion have
not been made. All that can be said is that there is no actual loss of N2 between the
time when the pupae form and the time when the flies emerge. It remains a possibility that nitrogenous waste products accumulate in the meconium which is passed
some hours after pupation. The N 2 content of the meconium is somewhat difficult
to estimate with accuracy, but it appears to be very low and almost negligible in
comparison with the total N2 content of the pupae. The indications are that there
is no excretion of N 2 into the meconium at all commensurate in amount with the
magnitude of the pupal respiration. Therefore, if there is a protein respiratory
metabolism at any time during the pupal period, it is of such a nature that the
nitrogenous 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 period
are most readily explained on the theory that some constituent of the tissues is
only partially oxidised and remains, in part at least, as a permanent constituent of
the body instead of being oxidised to products such as CO2 and H2O which are
eliminated.
2. There is definite evidence of a very marked synthesis of glucose during
pupation. If this glucose were all oxidised to CO2 and H2O, the expected Respiratory
Quotient would be about 0-7 or o-8 depending on whether the glucose is formed
from protein or fat. Actually the Respiratory Quotient seldom rises as high as 0*7
and 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; some
must almost certainly be used in building up the growing imaginal tissue substance.
3. The synthetic processes which form glucose are active throughout the whole
pupal period, though not uniformly so. The alcohol-ether soluble constituents of
the 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. There
is no storage (in the body of mature larvae) of glycogen or any other higher carbohydrate convertible by acid hydrolysis into glucose (or other reducing sugar).
Therefore, during the earlier part of the pupal period the glucose must be derived
from a protein source. The protein is used in such a way that no nitrogen is lost
by the body.
218
J.
G.
H.
FREW
4. During the latter half of the pupal period the fat content of the body
continually diminishes and during this period there may be a fat to carbohydrate
metabolism. The shape of the respiration curves shows a gradual diminution of
respiration during the early part of the pupal period and a gradual increase during
the latter half.
5. There is no reliable evidence which indicates that the metabolic processes
of the two sexes are different from each other.
REFERENCES.
STEPHENSON,
M. AND WHETHAM, M. D. (1925). Proc. Roy. Soc. B, 9 5 , 200.