J. Exp. Biol. (1969), 51, 99-105
Printed in Great Britain
QQ
PERMEABILITY TO WATER
OF THE SHELL OF THE EGG OF LOCUST A MIGRATORIA
MIGRATORIOIDES, WITH OBSERVATIONS ON THE
EGG OF TELEOGRYLLUS COMMODUS
BY T. O. BROWNING
Laboratoire de Biologie Animale, FaculU des Sciences,
Universite" de Paris and
Department of Entomology, Waite Agricultural Research Institute,
University of Adelaide
{Received 14 November 1968)
INTRODUCTION
Browning & Forrest (i960) used solutions of deuterium oxide in an attempt to
estimate the permeability to water of the shell of the egg of the cricket Teleogryllus
commodus. Their results were criticized by McFarlane (1966) on three main grounds:
first, that the deuterium oxide recovered could all have been contained in the shell
and thus no evidence of penetration into the contents of the shell was obtained;
secondly, and perhaps more cogently, that the deuterium oxide, if it were present
within the shell, could be the result of ionic exchange through the shell of deuterium
for hydrogen from the inside, and thus the results would provide no evidence of
permeability to deuterium oxide, and so of permeability to water; and thirdly, that
the charge on the layers of the shell might produce an ' imbibition pressure' in the
protein sufficient to prevent the diffusion of water through the shell even though no
'impermeable' barrier existed. If we are to understand the mechanism by which
insect eggs control the uptake of water, it is necessary to obtain a more direct observation of the permeability of the shell, and this paper is an account of experiments
designed to obtain such information.
The shells of eggs that imbibe water during their development are surely permeable
to water at the stage of imbibition. It is unlikely that they are also permeable to
large molecules, and thus they should behave as semi-permeable membranes towards
solutions of large molecules, at least during imbibition. The egg of Locusta migratoria
migratorioides is large (6—7 mg. when laid), and it is easy to cut off one end, remove
the embryo and yolk, fill the empty shell with water or with an osmotic solution,
and then ligature the open end to form a balloon. The balloon can then be immersed
in aqueous solutions of various osmotic potentials, and the behaviour of the balloon
observed. Experiments of this kind give information on the relative permeability of
the eggs, but no estimate of absolute permeability nor of flow-rate through the shell
can be obtained, because it is not feasible to weigh the balloons sufficiently accurately,
nor to measure their volume. The experiments described here were carried out with
the eggs of L. m. migratorioides and were designed to estimate the relative permeability
of the shell at different stages in the development of the egg. Eggs of diapausing
7-2
ioo
T . 0 . BROWNING
strains and non-diapausing strains were also compared. Confirmatory experiments
were also carried out with the much smaller eggs of Teleogryllus commodus (0-5-0-6 mg.
when laid).
METHODS
Eggs of L. m. migratorioid.es were obtained from cultures maintained by M. M.
Verdier, and incubated at 300 C. The day on which the eggs were laid was known,
but not the hour; there is thus an average error of + 12 hr. in the ages stated. Eggs
in which diapause development was complete had been stored for 6 months at 8° C.
Eggs were dissected from egg pods when required, the egg-pod remaining in the
damp sand of the oviposition vessel until it was needed. The eggs were immersed
immediately in water and bubbles adhering to, or emerging from, the porous shell
were gently removed with a fine brush. When preparing egg-shells for experiments,
the eggs were first punctured near the end with a fine needle and that end was then
snipped off with scissors. The contents of the shell were removed with a pipette
whose end had been smoothed in a flame. Balloons were ligatured using nylon
thread that was thick enough not to cut the shell. When filling balloons with an
osmotic solution the knot was pulled partly tight and then the shell was gently
squeezed with forceps to expel some of the solution. The knot was then tightened
and a flaccid balloon resulted. Balloons filled with water were tied off without
manipulation and they were usually turgid.
The osmotically active molecules employed were glucose (M.W. ca. 180) or polyethylene glycol (average M.W. 200). No differences were found in the behaviour of
these two substances. In most experiments a concentration of 2 M/1. was used, since
a preliminary experiment showed that the results were similar over the range 2 M/1.250 mM/L, except that at the lower concentrations the time required for observation
was much increased.
The strains of locusts used were: non-diapausing—Castel Sardo (Italy), Palacios y
Villefranca (Spain); diapausing—Kazalinsk (Russia), Magadino (Switzerland) and
Audenge (France). The crickets were a strain obtained from Canberra (Australia)
and maintained by Mile G. Cousin.
RESULTS
Locusta
(i) Experiments with balloons
The eggs of L. m. migratorioides, when incubated at 30° C , start to absorb water
rapidly after 5 days and complete absorption on the sixth day (Browning, 1968;
Verdier, 1957). If the egg is destined for diapause it enters diapause at the end of
anatrepsis and after water absorption is complete.
Balloons were made with the shells of (a) 'young' eggs, 1-2 days old at 300 C ,
(6) eggs 5-6 days old that were absorbing water rapidly, (c) eggs 7-8 days old that
had completed water absorption, (d) eggs in diapause that had been left at 300 C. for
10-12 days to ensure that they were firmly in diapause, and finally (e) eggs that had
completed their diapause development at low temperature.
The behaviour of shells from which the hydropyle end had been removed was
compared with shells in which the hydropyle end was left intact.
I.
Loss and gain of water by balloons made of empty egg-shells of Locusta migratoria migratorioides (for further explana'tion see text)
=
=
2.
Table
{
I
o
=
-
Days
Some eggs split and leaked on second day.
6.2 4.9*
.
7.3 5.9*
.
14.3 9.2.
.
14.7 14.4 13.6
(A) Weight of eggs on dry filter paper at 50 % R . H . and 30" C.
( B ) Diupause eggs re-moistened after 14 days
The solution used was glucose, 2 ~ / 1 .
Symbols
o no change; t slight increase in size; f rounded out; $ tightly turgid; - slight
decrease in size, surface partly concave; = almost flattened; G absolutely flat.
Non-diapause
Young eggs
Diapause
{Non-diapause
Fully imbibed eggs Diapause
§
+
Diapause completed
-
-
=
=
=
$
5
=
1
5
§
5
4
3
Age
Young
Imbibing
Fully imbibed
r
A
7
I
A
\
I
>
Eggs of diapausing or nondiapausing strain ...
... Non-diapause
Diapause
Non-diapause
Diapause
Non-diapause
Diapause
Hydropyle end included or
-tH
-H
+H
-H
+H
-H
+H
-H
+H
-H
+H
-H
removed
, - , A A - A
Days
Balloon containing water or
... ... ... H,O Soln H,O Soln H,O Soln H,O S o h H,O S o h H,O S o h H,O Soln H,O Soln H,O Soln H,O Soln H,O Soln H,O S o h after
solutionf
immersion
Days after balloons immersed
Table
0"
2
3
%
9
102
T. 0 .
BROWNING
In most experiments four kinds of balloons were compared; namely, (i) shells
with the hydropyle end were compared with (ii) shells without the hydropyle end,
and (iii) shells containing water and immersed in an osmotic solution were compared
with (iv) shells containing an osmotic solution and immersed in water. Two or three
eggs were used for each treatment. Very few balloons were spoiled, and ambiguous
results indicating a possible leak in a balloon seldom occurred.
Balloons filled with an osmotic solution absorbed water. They were slightly flaccid
when immersed in water and always sank to the bottom of the container. Gradually
the dents in the shell filled out until it had regained its rounded shape and then the
shoulders of the balloon at the ligature swelled until they were tightly rounded.
Such balloons, when squeezed lightly, returned instantly to their rounded state.
Balloons filled with water lost water to the solution on which they floated and only
sank into the solution when they were almost empty. They were usually fairly
turgid at the beginning and gradually shrank until only a small amount of liquid
remained in the rounded end. Often they became absolutely flat, often triangular in
cross-section.
The results of the experiments are shown in Table i. It can be seen that in all
cases the balloons either lost or gained water; under the conditions of the experiments
the shells were always permeable to water moving in both directions. There was no
evidence of permeability to solute molecules. Shells of young eggs and of eggs in
the process of absorbing water behaved in essentially the same way whether or not
they were destined for diapause. The shells of non-diapause, fully imbibed eggs also
behaved in the same way. It can also be seen that there was little difference between
shells with or without the hydropylar end.
On the other hand, the shells of eggs which had absorbed water and in which the
embryo had entered diapause were very different. They both lost and gained water
much more slowly than the other categories of eggs. Shells of eggs in which diapause
was complete were more permeable than those in diapause but less so than the other
categories.
(ii) Desiccation experiments
As another, though probably very different, measure of permeability, eggs were
exposed to unsaturated air and the loss of weight was followed by weighing individual
eggs at intervals. The results are shown in Table 2, when it is clear that young eggs
and non-diapausing, fully imbibed eggs lost water very rapidly to air at 50% relative
humidity and 300 C. Furthermore, many of these eggs, when observed on the second
day of desiccation, were found to have split open and leaked badly. Those that did
not split did not change significantly in weight when re-moistened. Diapausing eggs,
on the other hand, lost water very slowly under the conditions of the experiment,
and, when re-moistened after 14 days, absorbed water until their weight was even
greater than it had been before desiccation began. These experiments may be taken
as confirming the experiments with the balloons; that is to say that the permeability
of the shells of diapausing eggs is much lower than that of the other categories, that
the shells of diapausing eggs are, nevertheless, permeable to water moving in both
directions, and that the shells of the other categories of eggs used are highly permeable
to water moving out of the egg.
Permeability to water of egg shells
103
Teleogryllus
Eggs of T. commodus, i-day-old at 300 C , which therefore had not commenced
to absorb water (Browning, 1953), were made into balloons using a fine nylon thread.
Three concentrations of glucose solution were used, namely, 1 M/1., 500 mM/1. and
250 mM/1. The results are shown in Table 3. In all instances the balloons lost water
to the glucose solutions, or gained water if they contained glucose, but the rate of
loss was rather slow in the lower concentrations of glucose. The most striking difference
between this experiment and the experiments with locust egg shells, however, was
the fact that when the balloons contained the two higher concentrations of glucose
solution they not only became turgid, but the shell stretched, so that the volume of
the balloon became much larger than the volume of the corresponding part of the
eggTable 3. Loss and gain of water by balloons made of empty
egg shells of Teleogryllus commodus
Concentration of
glucose
Balloon containing water or
solution
Days after immersion
1
4
M
1
0-5 M
*
1
HaO
Soln
=
=
1
H2O
§
||
0-25 M
*
\
Soln
=
1
*
HaO
§
||
-
\
Soln
§
§
Symbols as in Table 1 except: || shell stretched beyond initial size.
DISCUSSION
The experiments reported above leave little doubt that the shells of L. m. migratorioides, and also probably those of T. commodus are permeable to water at all stages
of their development. With the methods used the only differences in permeability
that could be detected were between those eggs of L. m. migratorioides that had
entered diapause and those that had not; diapausing eggs became much less permeable
than they had been previously. The eggs of both Locusta (Browning, 1968; Verdier,
1957) and Teleogryllus (Browning, 1953; McFarlane & Furneaux, 1964) absorb
water only during a specific short period of their development. There must therefore
be some mechanism, as yet not understood, for preventing the net entry of water
by osmosis at other times. In all these experiments the cellular material within the
eggs was removed. It is likely that the control of water absorption is mediated through
the cells of the developing egg. Nevertheless, before a proper understanding of the
control of the movement of water in eggs is possible, the permeability of the shell
itself must be known.
The osmotic pressure of the solutions used in these experiments was often very
high, relative to the probable osmotic pressure of the tissues within the egg, which
is probably about 500-300 mM/1. (Grellet, 1966; Laughlin, 1957). Nevertheless, the
shells of the eggs of L. m. migratorioides did not stretch; they were thus capable of
sustaining the tension developed within the egg as the internal hydrostatic pressure
rose to equal and oppose the osmotic pressure. It is not possible to estimate the
104
T . 0 . BROWNING
actual osmotic gradient, since the solution within the shell became diluted as an
unknown amount of water flowed in. The shells of Teleogryllus are more plastic or
elastic and stretched under high pressures, but they did not burst.
McFarlane (1966) on the basis of experiments in which he removed water from
the eggs of crickets by floating them on concentrated solutions of sucrose, concluded
that an effective barrier to the movement of water through the shell of young eggs
exists and that it is probably due to the imbibition pressure of the protein of the shell
resisting the osmotic gradient. Such an imbibition pressure should be present in
the balloons made of young eggs of T. commodus, filled with 0-25 M glucose solution
and immersed in water (Table 3). Yet these balloons gained water rapidly. It appears
rather unlikely that the imbibition pressure can be adduced as the reason why young
eggs do not absorb water.
In L. m. migratorioides the whole of the egg shell was permeable to water, not
only the hydropylar end, as Slifer (1938) thought was true of Melanoplus differentialis.
The function of the hydropyle in the eggs of L. m. migratorioides remains obscure.
The experiments reported here confirm the conclusion reached by Browning and
Forrest (i960) that the shells of eggs that absorb water during their development are
permeable to water at all times, and they make the objections raised by McFarlane
(1966) seem less tenable. It now seems reasonable, provided adequate allowances
are made for ionic migration, to make use of isotopic water to obtain estimates or
the absolute permeability of eggs to water.
SUMMARY
1. Eggs of Locusta migratoria migratorioides were cut open at one end, the tissues
were removed, and the empty egg shells were filled with water or with an osmotic
solution. The open end was tied off to form a balloon. Balloons containing water
were placed in osmotic solutions, and those containing a solution were immersed in
water.
2. Balloons were made from eggs 1 or 2 days old before water absorption had
begun, from eggs during the course of rapid water absorption and from eggs in
which water absorption was complete. Diapausing and non-diapausing eggs were
used. The hydropylar end of the egg was included in the balloons in half the
observations.
3. All balloons containing solution gained water and all those containing water
lost it. Shells from diapausing eggs were much less permeable than all other categories.
Little difference was observed between balloons whether the hydropylar end was
present or not.
4. Eggs exposed to unsaturated air lost water rapidly, except those in diapause.
5. Balloons made with the shells of young eggs of Teleogryllus commodus were
also permeable to water in both directions.
6. The results are discussed in the light of their contribution to an understanding
of the control of the absorption of water in insect eggs.
This work was done during my tenure of a post as ' professeur d'echange' in the
Faculte" des Sciences, University de Paris. I wish to thank Mile le professeur Germaine
Cousin for her generous hospitality and encouragement while I worked in her
Permeability to water of egg shells
105
laboratory. M. M. Verdier provided the locust eggs. I am grateful to him for that,
and especially for many useful discussions and for his friendly help during my stay
in Paris.
REFERENCES
BROWNING, T. O. (1953). The influence of temperature and moisture on the uptake and loss of water
in the eggs of Gryllulus commodus Walker (Orthoptera: Gryllidae). J. exp. Biol. 30, 104-13.
BROWNING, T. O. (1968). Unpublished observations.
BROWNING, T. O. & FORREST, W. W. (i960). The permeability of the shell of the egg of Acheta commodus
Walker (Orthoptera: Gryllidae). J. exp. Biol. 37, 213-17.
GRELLET, P. (1966). Tenure en eau, pression osmotique et pH de l'ceuf de Scapsipedus marginatus
Afz. et Br. (Orthoptere: Gryllide). Application a la culture in vitro d'embryons. C.R. Acad. Sci.
Paris D 262, 1568-71.
LAUGHLIN, R. (1957). Absorption of water by the egg of the garden chafer, Phyllopertha horticola L.
J. exp. Biol. 34, 226-36.
MCFARLANE, J. E. (1966). The permeability of the cricket eggshell to water. J. Insect Physiol. 12,
1567-75.
MCFARLANE, J. E. & FURNEAUX, P. J. S. (1964). Revised curves for water absorption by the eggs of
the house cricket Acheta domesticus (L.) Can. J. Zool. 42, 239-43.
SLIFER, E. H. (1938). Formation and structure of a special water absorbing area in the membranes
covering the grasshopper egg. Q. J. microsc. Sci. 80, 437-547.
VERDIER, M. (1957). Observations sur la transmission pcear l'oeuf des caracteres phasaires chez Locusta
migratoria migratorioides R. & F. Mimeographed Report 3-erne Congres Union Internationale pour
l'Etude des Insectes Sociaux.