J. Exp. Biol. (1969), 51, 397-4°5
Printed in Great Britain
397
THE PERMEABILITY OF
THE SHELL OF THE EGG OF TELEOGRYLLUS
COMMODUS MEASURED WITH THE AID OF
TRITIATED WATER
BY T. O. BROWNING
Waite Agricultural Research Institute,
University of Adelaide
{Received 11 February 1969)
INTRODUCTION
The eggs of Teleogrylhis commodus, like those of many other species of insects,
absorb water during their development. The water enters through the shell at a
specific stage in the egg's development, and there is an as yet unresolved problem as to
how the control of the flow of water is effected (Browning, 1967). Recently it has been
shown that the isolated egg-shell of Locusta migratoria migratorioides is permeable to
water at all stages in the egg's development, and the same is probably true of T. commodus
(Browning, 1968.) The objections raised by McFarlane (1966) to earlier work by
Browning & Forrest (1960), using deuterated water to assess permeability, are
therefore probably not valid. Nevertheless Browning & Forrest (i960), for technical
reasons, were unable to separate the water held in the shell from that which had
penetrated to the yolk and embryo, and as McFarlane (1966) has pointed out, this
limits the conclusions that can be drawn from their work. The present experiment,
using tritiated water, which can be detected in much lower concentrations than was
possible with deuterated water and the equipment then available, aims to obtain
information on the following points: (1) the relative permeability of the shell at times
when little or no net flow of water is occurring, compared with the permeability
during the period of rapid water uptake; (2) the effect of the metabolic rate of the egg
on permeability; and (3) the relative quantities of water entering the shell and entering
the contents of the egg (using the word 'contents' to include the embryo, embryonic
fluids and the yolk).
METHODS AND MATERIALS
Eggs were obtained overnight from a large culture of crickets and placed at 120 C.
for 3 weeks, so that they would complete their diapause and develop promptly and
uniformly when incubated at 300 C. These eggs were then placed at 300 C. and each
day for the first 5 days five samples of 20 eggs each were taken from them. The time
spent at 300 C. before the samples were taken will be referred to as the 'pre-incubation
period'. Each sample was put in a Conway vessel on filter paper, and the paper was
moistened with enough tritium oxide solution (specific activity ioo/icml."1) to wet
the eggs and form a meniscus on their sides, without completely immersing them.
The lids were sealed on the vessels with grease and one vessel was placed at each of
398
T. O. BROWNING
five temperatures, namely 12, 17, 21, 28 and 300 C. Prior to the eggs being placed in
them, the vessels had been standing at their respective temperatures overnight, and
the eggs were transferred rapidly, so that they should reach the temperature of the
incubator as quickly as possible.
The temperatures at which the eggs remained while they were in contact with the
tritiated water were chosen to span the temperatures of 300 C , at which the eggs
develop rapidly, and 120 C , at which no appreciable morphogenesis occurs. On the
third day (that is, after 2 days pre-incubation at 30° C.) the eggs had begun to absorb
water, and they continued to increase in size and weight while they were in contact
with tritiated water at the higher temperatures, whereas there was no evidence of
continued water absorption at the lowest temperature (cf. weights of eggs given in
Table 1). Thus on the third day, at 300 C. the measurements represent the flow of
8
H g O* entering the egg along with a net influx of water, whereas at 120 C. they
represent the flow of 8H2O through a shell that was in the condition to absorb water,
but through which no net flow was occurring.
After the eggs had remained for 1, 6 and 24 hr. in contact with tritiated water, a
sample of five eggs was taken from each dish. The eggs were dried quickly on filter
paper, weighed and plunged into individual baths of liquid paraffin. Each egg was then
cut in half with scissors under the paraffin and the contents of the shell were sucked up
into a fine pipette. The contents of the pipette were ejected into 8 ml. of scintillation
fluid in a vial, the end of the pipette was rinsed several times in scintillation fluid by
sucking it up and down, and finally the end of the pipette was crushed against the
bottom of the vial, so that any material adhering to the glass remained in the vial.
The two pieces of the shell were transferred to another vial of scintillation fluid with
forceps. The vials from 1 day's observations were usually retained together; and the
activity of the 150 vials, together with the necessary blanks and standards, was
estimated in a Packard automatic scintillation spectrometer. Preliminary observations
had shown that no appreciable change in the apparent activity occurred when vials
were allowed to stand on the bench from between 10 min. and 2 days. No quenching
effect of the small quantities of paraffin used could be detected.
The contents of the eggs sometimes spilled as the eggs were cut in half. The yolk,
especially of young eggs, tended to spread on the glass of the vessel, under the paraffin,
and it was difficult to collect it all. Another problem encountered with young eggs was
that the shell sometimes crumpled as the contents were sucked out. It was then not
possible to suck up the whole contents; some remained inside the shell. If the problem was severe, the egg was rejected and a new one obtained, or a blank left in the
results. These difficulties no doubt accounted for some of the variability in the results.
There is also reason to suppose that some eggs had damaged shells, although each egg
was inspected when it was weighed and obviously damaged ones rejected.
RESULTS
Table 1 sets out the average quantity oftritiated water of specific activity ioo/icml." 1
recovered from both the contents and shells of the eggs on each of the 5 days and at
• 3HjO is used here, for the sake of brevity, to include *H|O and *HHO, and always refers to a solution
of specific activity
Egg of Teleogryllus commodus
399
each sampling time. The table also includes the mean fresh weight of the group of five
eggs from which the results were obtained. It is clear that results were highly variable,
but a statistical analysis of all the figures showed highly significant differences between
temperatures, sampling times and days of incubation. The results for contents and
shells were analysed separately because it was clear from the outset that the contents
contained significantly more radioactivity than did the shells under all circumstances,
on the average over five times as much. There is no doubt that the tritium oxide
penetrated rapidly through the shells and entered the contents of the eggs.
Table 1. Mean quantity (m/il) of3HtO* recovered from eggs
Hours immersion in tritiated water
6
I
A
Days
pre incuMean
bation Temp. wt.
at 30 0 C CO (m«-) Contents
0
25-7
9-8
36
115
2-2
8-3
i'3
067
802
184
0-64
0-64
0-67
0-64
0-69
0-70
39-8
47-5
n-8
8-2
066
7-8
o-68
0-69
0-64
0-65
0-64
1-2
41
0-7
063
063
063
91
4-8
7-8
3208
3619
180-4
57-7
39-3
371-3
54-4
54-3
49-7
io-6
8-6
215-2
289
402
17
2-5
1-7
0-9
12
0-64
0-67
o-6
30
25
084
085
55-o
37-1
8-i
089
o-88
21
0-85
17
082
090
35O
7-1
2O-2
3-7
76
i-5
50
o-8o
0-83
3°
21
[-21
•28
[•18
[-21
264-6
7 8-2
1687
95'6
I-O
30-6
95
078
[•29
176
[•30
[•13
10-4
[•27
12
•27
91
17
[-22
3°
44-2
94
5-6
[•24
3-3
[-28
[-23
17
Mean ratio
196
24
i-4
18
25
4
74'3
50-1
14-5
6-6
5'3
4-9
064
12
3
(mg.)
21
12
2
Shell
(mg-)
0-67
0-65
0-69
0-64
17
wt.
Contents
Shell
36-3
37
21
Mean
wt.
30
25
25
1
Mean
0 65
0-65
o-6o
0-64
0-64
o-66
0-64
30
24
25
•25
23
21
•25
286
146
17
•24
15-3
12
•22
II-2
3-4
9-7
Quantity •H,O in contents
5-O7 ±
Quantity *H,O in shell
[-22
•22
0-53
IO-I
2703
87-6
35-7
1648
139-8
56-8
61-9
25-8
5-66 ±
2-5
2-5
55-o
113
I-2I
I-OI
O-92
1694
131-0
596
34-6
176
1986
1892
1470
35'5
25-5
6194
6764
458-4
297-3
092
1810
•30
•26
•29
55°-4
133
60
•31
29-6
30-1
•29
.30
n-8
•31
107
6-i
•27
•31
O-22
Contents Shell
•26
4166
1569
1593
I4I-9
4986
357-4
252-6
125-7
999
283
228
133
59
43
480
368
279
7'7
5-8
861
974
55-i
54-i
32 9
99-8
716
48-1
281
20-9
825
61 8
49-1
230
19-5
551 ± 0 2 1
The results obtained after the eggs had been in 3HgO for only 1 hr. were more
variable than those obtained after longer exposures. This is also true of the ratio
between the quantity of water that had entered the contents and the quantity held in
the shell. These ratios, calculated for each sampling time are shown in Table i, and it
is clear that the ratio became fairly stable after 6 hr. immersion and the variability, as
expressed by the standard error, fell considerably. This probably reflects the variability in the shells of different eggs in their ability to hinder the passage of water into
26
Exp. Biol. 51, 2
400
T . 0 . BROWNING
and out of the egg, which may be expected to have a greater influence during the
initial period.
During the first 2 days the total quantity of water that penetrated the eggs was
small. The eggs were also small (about 0-65 mg.), and there was little change in
weight. On the third day, however (that is, after 2 days pre-incubation), the eggs had
all begun to absorb water (average weight about 0-85 mg.), and the total quantity of
tritiated water absorbed subsequently was greater. The eggs treated at 30 and 250 C.
completed their water absorption during the 24 hr. period, but in those treated at
Table 2. Meanflozo-rates(m/il. mm.^hrr1) through the shell
Days preincubation
at 30 0 C
0
1
2
Hours immersion in tritiated water
1
6
3-5
26
2-O
21
1-5
13
24
i-4
13
I-O
o-6
17
1-3
o-s
0-4
13
i-o
0-3
0-2
3°
8-8
25
32
i-6
0-9
19
19
21
09
0-4
i-4
17
o-6
o-5
o-3
Temp. (°C.)
30
25
13
0-4
0-4
03
3O
25
11-3
7-5
10-2
10-2
43
21
3-4
1-5
i-6
5-1
4-4
3-3
3'5
2-O
i-3
1-7
43
5-2
41
17
43-3
114
23-0
15-6
96
12
i-4
0-9
3-6
i-o
i-o
0-9
30
25
6-6
41
31
4-2
36
2-2
21
2-2
17
2-3
1-7
1-4
i-6
07
i-5
08
17
12
3
3°
25
21
4
12
2-2
o-6
lower temperatures, and in which the rate of water absorption was appreciably slower
(no change in weight could be detected at 17 or 120 C ) , much less radioactivity was
found in the eggs. Nevertheless, even the eggs that had gained no weight while exposed
to tritiated water had still gained appreciable radioactivity. On the last 2 days of the
experiment, when the eggs had already completed their water absorption during the
period of pre-incubation at 300 C , large quantities of SH2O were found in all eggs, and
again the quantity detected depended on the temperature at which the eggs were held
in tritiated water.
The figures in Table 1 are simply the gross quantities of 3HgO recovered, calculated
from the counts obtained, and give little information on the permeability of the shell,
because its area changes considerably during the period of water absorption. In
Table 2 the flow-rates through the shell are shown. These figures have been calculated
from the quantitity of 3H2O recovered from the contents of the eggs after each timeinterval and take into account the area of the shell of each egg (see Appendix). In
Egg of Teleogryllus commodus
401
general the rates of flow were high during the first 6 hr., but fell considerably when the
whole 24 hr. period was considered, and as the concentration of 8H2O inside the shell
became more nearly equal to that outside (see Table 3). There were no significant
differences detectable between the over-all flow-rates into eggs that had not been preincubated compared with those pre-incubated for only 1 day, nor among those preincubated for 2, 3 or 4 days, but the rates after the short pre-incubation times were
significantly lower (P = 0-05) than those after the longer times. It may be concluded
that the shell is rather less permeable during the first two days than it is subsequently.
Table 3. Mean proportion (per cent) of water replaced by bathing solution
Days pre1x1 c u u u u u n
at 30 0 C
0
Hours immersion in triuated water
Temp. (°C)
3°
25
21
17
1
2
24
6i-8
470
219
130
6-3
683
i-5
31
3°
14-3
293
»5
2-7
17
3-o
i-o
'39
89
680
21
131
12
07
4-i
4-1
30
25
I2'O
6-2
29
i-8
i-8
17
5i-8
IO-2
622
84-2
73-8
842
510
8o- S
59o
242
93
342
30
25
4O-5
522
10-3
246
657
497
21
214
17
12-2
12
1*1
3°
6-i
25
3-7
19-7
io-o
4-4
59
36
21
1-9
7-0
17
19
4 0
77
3-5
12
4
2-2
19
6
287
19-0
95
5'3
12
21
3
1
5-2
2-4
13
2I-O
188
173
566
44-7
30-3
15-4
12-2
Table 3 shows the proportion of the total water in the egg that was replaced by
exchange from the outside during each of the sampling periods. These values are for
the whole egg, because it was not found practicable to estimate the proportion of
water in the shell and its contents separately. To estimate the proportion of water in
whole eggs, the eggs were weighed individually on a torsion balance of 2 mg. capacity,
reading to o-ooi mg., dried for 24 hr. at 1050 C, and weighed again quickly on the
same balance. The weight of water in each egg was then plotted against its fresh weight
and a line was fitted through the points by eye. Very little variation was found about
this line, which represented 250 eggs whose fresh weights varied from 0-52-1-40 mg.,
so that little error was involved in reading from this graph the weight of water contained
in an egg whose fresh weight was known. Taking the weight of each egg, and the
quantity of 8H2O in the whole egg, the proportion of water in the egg that had been
26-t
402
T . O. BROWNING
replaced by the bathing solution was calculated and the results are summarized in
Table 3. Only when the eggs had been pre-incubated for 2 days and were in the condition to absorb water, and only when placed in tritiated water at the higher temperatures at which water absorption actually occurred, was almost all the water in the egg
replaced in 24 hr. Otherwise, only about half the water at the higher temperatures,
and much less at the lower temperatures, was replaced during the 24 hr. of contact
with 3H2O.
DISCUSSION
The results in Table 1 show that 3H2O penetrates the shell of the egg of Teleogryllus
commodus rapidly, a small proportion remaining in the shell, but most accumulating
in the internal tissues. The isolated egg-shell has been shown to be permeable to
water when it is used as an osmometer membrane (Browning, 1968), and it thus
appears likely that, in this experiment, much of the water penetrated the shell by
mass flow, rather than by ionic exchange, as was postulated by McFarlane (1966).
In the section on Methods the great variability found within groups of eggs
treated similarly, and between groups, was mentioned, and some of the probable
reasons for this were stated. However, it is possible that some of the variability is
biological, rather than due to experimental technique, and may reflect the variable
structure of the shells of different eggs. Phillips & Dockrill (1968) found a similar
variability in the permeability to larger molecules of a cuticular structure in Schistocerca
gregaria. Such variability may appear more likely if the function of the shell is primarily
to provide a strong container for the tissues within, rather than to act as an impermeable barrier from the environment This question will be discussed later.
A marked difference was found in the amount of water that entered the eggs at
different temperatures. Over-all the average temperature coefficient relating the flowrate to temperature (Q10) was about 3 when allowance is made for highly aberrant
values (for example after 1 hr. on day 3 in Table 2). It is perhaps unlikely that such a
high value would be due simply to the increased rate of osmoticflowas the temperature
increased; rather, some metabolic phenomenon seems to be implicated. All eggs on
any day were at the same stage of development when they were placed in 8 H a O, all
having spent the same time developing at 300 C. However, those that were placed in
3
H2O at 300 C. continued their development without interruption, whereas the
development of all the others was retarded at the lower temperatures. For example at
300 C. the egg requires 2 days to reach the stage at which water is absorbed, and
absorption occurs in about 12 hr., whereas at 120 C. over 30 days are required to
reach the stage of water absorption and absorption occupies another 12 days. Thus the
general metabolism of the eggs is greatly slowed down at lower temperatures, and the
concurrent slowing in the rate offlowof water through the shell may well be due to the
rate at which water, once inside the shell, is transported through the egg. Such
transport can be thought of as a kind of biological stirring mechanism, which would
operate much more slowly at 12 than at 300 C.
At 30° C. the rate of transfer through the shell, as measured by the amount of
tritiated water taken up by the egg, is sufficient to allow the egg to absorb all the water
it requires during morphogenesis in about 12 hr. Even at the lowest rate of flow
observed during the period when the eggs had begun to absorb water (i.e. at 120 C. on
Egg of Teleogryllus commodus
403
the third day), the amount of water required could flow through the shell in less than
3 days, whereas some 12 days are needed for complete absorption at this temperature.
At least at the lower temperatures, some mechanism other than the permeability of the
shell must be restricting the entry of water.
It has been shown that anoxia during the period of water absorption stops the
further flow of water into the egg of crickets (Browning, 1965; McFarlane & Kennard,
i960). Lack of oxygen would not be expected to interfere with osmotic flow, but it
would be expected to stop the growth of the egg. Both McFarlane (1963) and Browning
(1967) have shown that a profound change occurs in the endochorion of the shell just
before water absorption begins. If this change is thought of as a growth process,
causing the shell to become greater in area (rather than as signalling only a change in
permeability, as McFarlane thought), an increasedflowof water into the egg would be
expected, and lack of oxygen would be expected to prevent continued absorption. Both
of these conditions are observed. This may not be a general explanation for water
absorption, since some eggs probably continue to absorb water even under anoxic
conditions (Dr A. D. Lees, personal communication; T. O. Browning, unpublished
observations).
If we can conclude that the shell of the egg is indeed permeable to water at all
stages during its development, the question arises why the egg does not absorb water
during the first few days of its development, nor during the period it spends at low
temperature prior to incubation. During the early part of development the osmotic
potential of the solution within the egg is likely to be greater than it is after water has
been absorbed and dilution occurs (Grellet, 1966; Laughlin, 1957). It has been shown
that when the shell of the young egg is made into a balloon partly filled with a solution
of glucose equivalent in concentration to the solution within the egg, and the balloon is
immersed in water, it absorbs water but the shell does not stretch or split (Browning,
1968). Under these circumstances a sufficient hydrostatic pressure must build up within
the shell to oppose the net entry of water by osmosis. Perhaps a similar mechanism
operates in the living egg.
SUMMARY
1. Eggs of Teleogryllus commodus were incubated at 300 C. for 5 days; that is, during
the initial 2 days when little change in weight is observed, the third day when the eggs
absorb water rapidly, and during 2 more days when little further change in weight is
seen.
2. Each day samples of eggs were immersed in tritiated water (100 /jcml."1) at 30,
25, 21, 17 and 120 C. After treatment for 1, 6 and 24 hr. the radioactivity in the shells
and in the contents of five eggs from each group was measured.
3. About five times as much radioactivity was found in the contents as in the shells.
The activity was greatest at 300 C. and least at 120 C. on all days and at all times.
Radioactivity taken up by the eggs was least on the first 2 days, rose sharply on the
third day and remained high thereafter.
4. The results are discussed in relation to the mechanism of water-absorption in the
eggs.
404
T. O. BROWNING
I wish to thank Miss Meredith Porter, B.Ag.Sc, for her able assistance with this
work. Financial support from the Australian Research Grants Committee is gratefully
acknowledged.
APPENDIX
Method used to estimate area of egg-shells
A large group of eggs was incubated at 30° C. and from these 10 eggs were taken
each day. Each egg was weighed and placed on a glass slide and its outline was traced
using a camera Uicida and a low-power compound microscope with reflected light.
The image of a graticule 2 mm. in length and divided into 200 parts was then traced
on to a strip of celluloid and this was used to measure the egg using its outline. Measurements were made to the nearest o-oi mm. The method was as follows. A line representing the longest axis of the egg was ruled on each tracing and, assuming the egg was
circular in cross section, the diameter of the egg was measured at regular intervals
along the axis at right angles. The object was to cut the egg into a series of thin slices of
equal thickness, each of which could be regarded as a flat cylinder, except the two end
ones. One of the latter had a thickness equal to that of the slices, and the other was the
residual piece after all slices of predetermined thickness had been cut. Its thickness
was either equal to or less than that of all the others.
Treating all the slices except the first and last as cylinders whose height was known
and whose diameter was the mean of the diameters at each end, the area of the curved
surface was calculated. The two end slices were treated as segments of spheres whose
depths and the diameters of whose bases were measured, and from these measurements the areas of the curved surfaces were calculated. The total area of the shell was
taken as the sum of the areas of all the curved surfaces.
Probably the main source of error in this procedure lies in treating the slices as
cylinders. This is particularly true near the ends where the diameter is changing
rapidly. To estimate the error likely to be involved in the method, the outline of one
egg was transected to give 81 slices, as many as could be accurately spaced along the
axis, and the area was calculated from these. The same outline was again transected
using slices 5 times and 10 times the thickness of the original one. This produced 17
and 9 slices respectively. The surface area was again calculated on these two bases.
Assuming that the area calculated using 81 slices was correct, the error involved in
using 17 slices was +2-4% and with only 9 slices it was +5*2%. Since the error
involved in using the intermediate thickness of slice was only about 2-3 % of the best
estimate obtained with five times as much work, the intermediate thickness was used to
estimate the surface areas of the shells of 10 eggs on each day of incubation at 300 C.
The weight of each egg was then plotted against its calculated surface area, and a
straight line was ruled through the points by eye. The variability about this line was
very small. The line was then used to read off the surface area of eggs whose weight
was known.
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