The Penetration of Some Non-Polar Molecules in Solution Through the Egg-Shells of Locusta Migratoria Migratorioides and Teleogryllus Commodus

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.

Eggs of L. m. migratoria 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.

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 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.

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 ¹⁴C-labelled solute to produce a specific activity of 1μ¹⁴C_i 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 0·8 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 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.

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 μCi 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).

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 × 10 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. l^-1, then this rate of loss would result in all the small molecules passing out through the shell in less than 1 month.

McFarlane (1960) 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 (1971a). These changes, seen in ultramicrographs, probably correspond to the changes that can be seen in surface view at this time (McFarlane, 1960, 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, 1969b; Grellet, 1971a) or to other molecules (Table 2).

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, 1971b).

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.