J. Exp. Biol. (i97»). 56, 769-773
With 1 text-figure
printed in Great Britain
769
THE PENETRATION OF SOME
NON-POLAR MOLECULES IN SOLUTION THROUGH THE
EGG-SHELLS OF LOCUSTA MIGRATORIA
MIGRATORIOIDES AND TELEOGRYLLUS COMMODUS
BY T. O. BROWNING
Department of Entomology, Watte Agricultural Research Institute,
University of Adelaide
(Received 17 November 1971)
INTRODUCTION
The shells of the eggs of Locusta migratoria migratorioides and Teleogryllus commodus
have been shown to be permeable to water, even at periods during their development
and under conditions where no net flow of water across the shell could be detected
(Browning, 1969 a, b). Similar conclusions have recently been reached by Grellet
(1971 a) on the shell of the egg of Scapsipedus marginatus. Hogan (1962) showed that
when diapausing eggs of Teleogryllus commodus were soaked in solutions of urea,
their diapause was rapidly terminated, and urea passed through the shell. Phillips &
Dockrill (1968) have shown that a wide range of non-polar solutes pass through the
cuticular intima of the isolated rectum of Schistocerca gregaria. There is thus evidence
that not only water but also molecules in solution can diffuse through the dense
proteinaceous ' membranes' of insects, and this paper records the results of observations designed to measure the rate of passage of molecules of varying size through
egg-shells.
MATERIALS
Eggs of L. m. mtgratoria were obtained from a group of adults caught near Kyoto,
Japan. Eggs of T. commodus were obtained from cultures maintained in this laboratory.
The areas of the shells of crickets' eggs were estimated by the method described
previously (Browning, 19696). Methods used for each of the observations are described in the relevant section.
RESULTS
(i) Osmosis
Osmotic balloons were made by snipping the micropylar ends from eggs of L. m.
migratoria, sucking the contents from the larger part with a pipette and tying the cut
end off with fine thread. In this way a balloon could be filled with either a solution or
water (Browning, 1969a). The solutions used to fill the balloons, or in which balloons
filled with water were immersed, were of glucose, malonamide or urea, all at a concentration of 0-5 mol.l" 1 . The results (Table 1) show that, with glucose, these egg-shells
behaved in essentially the same way as those of L. m. migratorioides and T. commodus;
those that were filled with glucose solution and immersed in water swelled and became
770
T. O. BROWNING
Table i. Appearance of osmotic balloons at intervals after immersion in fluid. Th
containing solution were indented when placed in water, whereas those containing water
were usually turgid when placed in solution
Appearance of balloons after:
(7 egg8 in each experiment)
Balloons containing:
Glucose
Malonamide
Urea
Balloons immersed in:
Glucose
Malonamide
Urea
1 day
2 days
5 T, 2 Sw
5 Sw, a N
2 Sw, 5 N
7T
6 Sw, 1 N
2 Sw, 5 N
6 F, 1 N
5 Sh, 2 N
7N
7F
5 Sh, 2 N
1 Sh, 6 N
T = turgid; Sw = swelling; Sh = shrinking; F = flattened; N = no change.
turgid, and those that were filled with water and immersed in solution became flattened. Water passed through the membrane according to the difference in osmotic
potential of the liquids on the two sides. However, little or no change was observed
when urea was used as the solute, and with malonamide the results were erratic. The
result suggests that the shell of these eggs may be relatively permeable to urea, and
much less so to the larger molecule of glucose. An attempt was therefore made to
measure the diffusion of molecules through egg-shells more directly.
(ii) Perfusion
Eggs of T. commodus were weighed, immersed in water, the micropylar end was
cut off with fine scissors, and the yolk and cellular structures were sucked out with
a pipette. The empty egg-shell was then attached to a perfusion apparatus, as shown
in Fig. 1. The egg-shell was perfused for at least 1 h with a solution adjusted to
0*5 mol.l" 1 . In most experiments the shell was immersed in distilled water; in others
it was immersed in a solution similar to the perfusing solution except that it was not
radioactive. After the perfusing solution had almost completely run through the
apparatus, an aqueous solution of eosin was run through the egg-shell for at least
half an hour. If dye was detected in the vial the experiment was discarded. In other
experiments, even though no leak was detected with the dye, the radioactivity in the
vial was so great that a leak was suspected and the results were discarded.
The solutions used were of glucose, ribose, acetamide and urea, and for perfusion
each of these was made radioactive by adding to the stock solution sufficient 14Clabelled solute to produce a specific activity of 1 /tCi ml"1. At the end of an experiment
the vial, containing 0-4 ml water or solution, was plunged into 8 ml scintillation fluid
and the radioactivity was measured in a Packard liquid scintillation spectrometer.
Eggs that had not begun, or were just beginning, to absorb water (weighing between
0-5 and 0-79 mg) and in which the serosal cuticle was absent, were recorded separately
from those which had begun or had completed water absorption (weighing between
o-8 and 1-3 mg) and in which the serosal cuticle was present.
The quantities of radioactive material recovered in the external solution were very
small (averaging 5 to 10 times the background) but the results shown in Table 2
Penetration of some non-polar molecules
1
B
C
D
A
771
\
0
Fig. 1. Perfusion apparatus made by sealing two pieces of Pyrex glass tube together at their
enda and pulling them out to a fine taper. One piece was then bent over and pulled out in the
other direction to form the effluent tube (E); the other served as the reservoir (A). Wax was
melted near the end and trimmed to fit the egg-shell (C) closely. The shell was tied firmly
around the waxed ends of the double glass tube with fine thread (B). The egg-shell was
immersed in a small vial (D) containing 0-4 ml fluid.
Table 2. Passage of molecules through the egg-shells of crickets.
Concentration of all solutions 0-5 mol. l~x, specific activity 1 fid ml~x
Weight
of eggs
(mg)
0-5-0-79
0-5-0-79
0-5-0-79
0-5-0-79
0-8-1-3
—
_
—
o-6-o-8
Equiv.
hydra ted
molecular
Perfusing
solution
Glucose
Ribose
Acetamide
Urea
Glucose
Ribose
Acetamide
Urea
Glucose
Urea
radius
(A)
4-ao
360
3-37
3-03
Bathing
solution
Water
Water
Water
Water
Water
Water
Water
Water
Glucose
Urea
No. of
observations
4
7
11
17
S
9
7
9
3
5
Rate of passage
(mol. mm-*
h"1 X io-«)
0-5 ±o-1
o-7±o-i
10-3 ±3-6
10-7 ±1-9
o-6±o-3
o-4±o-j
3-5 ±0-3
3-8 ±0-4
3-4 ±0-4
14-7 ±4-5
indicate a real permeability of the egg-shell to each of the molecules, and show that
the smaller molecules, urea and acetamide, passed through the shell more readily than
the larger ribose and glucose. The presence of the serosal cuticle significantly retarded
the flow of the smaller molecules through the shell. This probably holds true for the
larger molecules also, but the results were not significantly different between the two
kinds of eggs.
772
T. O. BROWNING
Table 3. Passage of molecules into developing eggs of crickets.
Concentration of all solutions 0*5 mol.lr1, specific activity 1 pCi rah1
Compound
Glucose
Ribose
Acetamide
Urea
No. of
Rate of passage
observations (mol mm" 1 h - 1 x io~*)
8
o-0O2±o-ooi
4
0-008 ± 0-005
8
o-l 10 ±0-050
5
0-050 ±0-030
(iii) Developing eggs
Whole eggs of T. commodus were allowed to stand at 30 °C for 24 h in sealed Conway
dishes on filter-paper wetted with radioactive solutions of specific activity 1 fid ml"1.
The eggs were then removed, rinsed quickly in three changes of distilled water, dried,
weighed and immersed in liquid paraffin. They were then cut in half with scissors and
the contents of the shell were removed with a Pasteur pipette and transferred to a vial
of scintillation fluid whose background radiation had been measured previously.
The end of the pipette was rinsed with scintillation fluid by sucking it up and down
several times and then the end of the pipette was crushed against the bottom of the
vial, leaving the broken glass inside. The solutions used were the same as in the perfusion experiments above.
The results of this experiment are shown in Table 3. No significant uptake could be
measured when eggs stood in solutions of glucose or ribose. Small but significant
amounts of radioactivity were found in eggs that had been bathed in acetamide and
urea, but the difference between these was not statistically significant. The rates of
penetration were much less than those found in the perfusion experiments (Table 2).
DISCUSSION
The results of all the experiments described here lead to the conclusion that the
shells of the eggs of the insects used are permeable to non-polar molecules even with
a diameter as large as that of glucose. Hogan (1962) provided evidence that urea
passed through the shells of cricket eggs and it is well known that water and other
substances (gases in solution and ions) can pass through such shells. Nevertheless, it is
unlikely that molecules in solution readily pass out of insect eggs in nature. The rate
of penetration observed in the perfusion experiments would result in the loss of
about 5 x icr 6 mole per egg during the time the egg spends in moist soil. If we
assume that the concentration of the solution in the egg is 0-5 mol.I"1, then this rate
of loss would result in all the small molecules passing out through the shell in less than
1 month.
McFarlane (i960) and Browning (1967) showed that the chorion of the eggs of
crickets undergoes considerable structural changes during the development of the
egg and this has been confirmed and extended by Furneaux, James & Potter (1969)
and Grellet (1971c). These changes, seen in ultramicrographs, probably correspond
to the changes that can be seen in surface view at this time (McFarlane, i960, 1965).
But such changes, which have the appearance of fractures in the endochorion, do not
result in any marked increase in the permeability of the shell either to water (Browning, 1969*; Grellet, 1971a) or to other molecules (Table 2).
Penetration of some non-polar molecules
773
Small molecules passed through the shell of living eggs much less rapidly than they
passed out through dead egg-shells, indicating the existence of a barrier to the movement of substances in solution that is associated with the integrity of the living
tissues within the egg. The cells of the serosa form a continuous layer beneath the
shell and it seems likely that they, or at least their outer cell membranes, control the
movement of substances into and out from the egg (Grellet, 1971 b).
Grellet (1971a) used the shells of eggs of Scapsipedus marginatus as osmometer
bladders which he filled with a solution of NaCl. Under these circumstances water
passed through the membrane, but he gives no indication that NaCl passed in the
other direction. The results obtained here would make that seem likely.
SUMMARY
1. The micropylar ends of eggs of Locusta migratoria migratoria were cut off, the
tissues were removed, and the empty egg-shells were filled with water or with a
solution. The open end was tied off to form a balloon. Balloons containing water were
placed in a solution, and those containing solution were placed in water.
2. Balloons containing glucose solution swelled in water and those placed in glucose
solution shrank. Little change was observed either in balloons containing urea or in
those placed in urea. Results with solutions of malonamide were erratic.
3. Empty egg-shells of Teleogryllus commodus were perfused with radioactive solutions of substances of varying hydrated molecular radii. The smaller molecules (acetamide and urea) passed through the shell into the surrounding liquid more rapidly
than the larger ones (glucose and ribose). Shells in which the serosal cuticle was absent
were more permeable than those in which it was present.
4. Molecules penetrated living eggs much less readily than they passed out
through dead egg-shells.
5. The results are discussed in relation to the permeability of the shells of insect
eggs to water and their capacity to restrict the leaching of molecules from the tissues
of the egg.
REFERENCES
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and J. E. Treherne. Edinburgh and London: Oliver and Boyd.
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BROWNING, T. O. (19696). The permeability of the shell of the egg of Teleogryllus commodus measured
with the aid of tritdated water. J. exp. Biol. 51, 397-4°SFURNBAUX, P. J. S., JAMES, C. R. & POTTER, S. A. (1969). The egg shell of the house cricket {Acheta
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