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The egg of Tetrix (Tetrigidae, Orthoptera), with a discussion on the probable significance of the anterior horn By J. C. HARTLEY (From the Department of Zoology, University of Bristol) Summary The structure of the egg-wall of Tetrix vittata and the changes that occur in it during development are described. The chorion has a hollow layer held open by struts and freely permeable to both air and water. At the posterior end the struts become finer and the chorion then resembles that of the Acrididae. A thick serosal cuticle develops with a hydropyle at the posterior end. This cuticle is dissolved before hatching. The egg takes up water and increases its volume during development. There is a long horn with a hollow core at the anterior end of the egg. This horn is entirely chorionic and appears to provide the egg with an expansion chamber.

Introduction APART from the work of Hancock (1902), the life-history of the Tetrigidae has received little attention. Hancock illustrated and described the appearance of the eggs and noted the behaviour of the female during oviposition. Specimens of Tetrix vittata Zett. and T. subulata L. were collected in April and kept in the laboratory. They were cultured in vivaria containing a layer of moist sand and pieces of short moss. Eggs were laid in clumps in cells excavated under the moss or in the sand. Egg-laying ceased by the middle of May and the adults began to die at this time. Development of the eggs takes 3 or 4 weeks, and hatching occurs by rupture of the egg-wall. During development the eggs of Tetrix, like those of the Acrididae, take up water and expand. Another feature shared by the acridids is the function of the amnio- serosal fluid in sealing small ruptures in the egg-wall. This was previously observed in the Acrididae by Jones (1958) and Hartley (1961).

Results Description of the egg The eggs of both species are very alike. The following account is based on those of T. vittata. The eggs have been described by Hancock (1902) as wine- bottle-shaped. They are more or less ellipsoidal, with an anterior horn (fig. 1, A). The egg, excluding the horn, is about 2-2 mm long and has a maximum diameter of about 0-62 mm initially. The horn is between o-6 and 0-8 mm long and 0-12 to 0-15 mm wide. Numerous micropyles are present in a band around the egg just posterior to the middle. They are funnel-shaped and run slightly posteriorly through the chorion. The chorion is 0-02 mm thick over the main body of the egg. It consists of a continuous basal layer 2-5 /x thick with buttressed struts arising from it [Quarterly Journal of Microscopical Science, Vol. 103, part 2, pp. 253-9, June 1962.] 254 Hartley—The egg of Tetrix (fig. i, B). The struts are also splayed out at their outer ends and form a per- forated surface sheet. There is a distinct and approximately hexagonal pattern visible in surface view, which is produced by some of the struts being ex- panded or linked to form partial walls corresponding to the boundaries of the follicular cells (fig. i, c).

FIG. I. A, egg of T. vittata showing covering of granular secretion, absent on one side which was in contact with another egg. B, section of chorion and secretion from side of egg. There is a thin serosal cuticle, c, surface view of chorion, showing cell pattern.

FIG. 2. Section of egg-wall at posterior quarter of egg. The chorion is a felt-like meshwork at the posterior pole. Beneath it is the yellow cuticle (thick black line) and at the right the hydropyle, with white cuticle and hydropyle cells beneath.

At the posterior pole of the egg the chorion has a different structure (fig. 2). The basal layer becomes thinner and is slightly less than half the thickness found elsewhere. The struts are replaced by a fine system of interlocking fibres producing a felt-like appearance. The inner half of this felt-like part of the chorion is rather denser than the outer part. This region of the chorion is therefore very like that of Chorthippus and some other Acrididae (Hartley, 1961). The horn of the egg is entirely chorion. The basal layer of the chorion is Hartley—The egg of Tetrix 255 thicker in this region and is irregularly star-shaped in cross-section. The central core is rather fragmentary and thus the horn has the appearance of a collapsed tube (fig. 3, A). The form of the outer layer of the chorion on the horn also differs from that on the sides of the egg. The struts are replaced by continuous walls forming a honeycomb arrangement of cells. These are

FIG. 3. The anterior horn. A, cross-section. B, longitudinal section of horn of newly-laid egg. c, longitudinal section of horn of developing egg. covered by a thin, porous layer of chorion. The height of the walls in this part varies according to the shape of the inner layer, so that the outer surface is evenly rounded. When the eggs are laid they are covered with a secretion that very effectively gums them together. The secretion has the appearance of a foam in which the bubbles are replaced by a granular substance (fig. 1, B). This secretion forms an additional, coarsely granular layer over those parts of the eggs that are exposed (fig. 1, A). During the later stages of development the chorion fragments as the egg expands. This phenomenon has been noted in the Acrididae (Hartley, 1961). In Tetrix not only the chorion but also the outer secretion cracks. At this time the secretion no longer holds the eggs firmly together and they can easily be separated. Another change in the chorion that occurs during development is in the horn. Longitudinal sections show that as the egg develops the serosal cuticle is pushed up into the core of the horn and expands it (fig. 3, B, c). The chorion in this part becomes stretched and thinner, so that it is more like the rest of the chorion on the sides of the egg. The chorion is readily wetted by water, and drops applied to the surface 256 Hartley—The egg of Tetrix will spread through it. There is less tendency for water to spread through the chorion of the horn, but air is not held against water. The outer secretion also absorbs water.

The serosal cuticle The development changes in the egg-wall are very like those found in the Acrididae, as described by Slifer (1937). A serosal cuticle is laid down beneath the chorion. This has the usual two layers, a yellow cuticle less than a micron thick, and a white cuticle about 12 /J, thick. This cuticle is produced before the egg starts to expand by uptake of water. A few days before hatching, the white cuticle is dissolved. This probably occurs in the same way as has been described for Melanoplus (Slifer, 1937), since a pair of very conspicuous pleuropodia are present at this time. Just before hatching the embryo drinks the fluid surrounding it.

The hydropyle Examination of the eggs, after removal of the chorion by concentrated sodium hypochlorite solution, revealed a circular patch about 180 /x in diameter at the posterior end with a different appearance to the rest of the yellow cuticle. Longitudinal sections through this area revealed a difference in the serosal cuticles (fig. 2). The yellow cuticle is replaced by a thicker membrane and beneath it the white cuticle is thinner than elsewhere on the egg. Attached to the inner side of the serosal cuticle in this place are some large cells, which persist during most of the development of the egg. This part of the serosal cuticle is much more firmly attached to the chorion than elsewhere. The structure therefore shows very great resemblance to the hydropyle described by Slifer (1938) in Melanoplus eggs. That this structure is a hydropyle was confirmed by testing, after removal of the chorion, with a solution of fast green, which entered at this place only. Similar results were obtained with fixatives and KOH solution. Also, eggs were unable to take up water after removal of the chorion unless the hydropyle was in direct contact with the water.

Change in volume Measurements were made on developing eggs to determine the change in volume. The results are expressed in table 1. The information in the table was based on camera lucida drawings of the eggs in side view. The volumes were calculated by the formula

where V is the volume, A the area of the drawing measured by a planimeter, and rt and r2 the distances of the centres of gravity of the longitudinal halves of the area from the longitudinal axis (see fig. 4). This is on the assumption that the eggs are circular in cross-section, which is approximately so. Hartley—The egg of Tetrix 257 The first line in the table is for the average of 5 eggs from freshly laid egg clumps. The difficulty of separating the eggs at this stage made it impossible to follow the developmental stages of these particular eggs. Eggs 2 to 6 were measured as soon as the eggs could be separated. Eggs 7 and 8 were fully swollen and measured shortly before hatching. Eggs 9 to 11 were measured

TABLE I

Length excluding Maximum Egg horn (L) width (W) Area {A) 2 Volume

i(X5) 218 0-62 [•03 0 13 044 2 2-2I 071 [•30 0 16 O'6s 3 2-21 071 1-2,1 0 IS 0-62 4 214 066 [•10 0 14 049 5 2-17 068 [•24 0 16 0-62 6 2-17 081 [-46 0 r7 078 7 2-25 084 [-59 0 18 095 8 2-36 086 •56 0 19 093 9<* 2-ZS 07s [•42 0 r7 076 9b z-48 0-90 75 0 20 I'lO 10a 2-25 079 •38 0 17 074 10b 2-40 0-82 •62 0 18 0-92 11a 2-14 0-64 •17 0 16 OS9 11b 2-25 °75 •41 0-18 080

FIG. 4. Diagram showing measurements of egg used in table i. twice, (a) as soon as they could be separated, and (b) when development was complete. From this table it can be seen that the eggs tend to double their volume during development. Similar increases have been recorded in the Acrididae (Roonwal, 1936; Slifer, 1938). Discussion The Tetrigidae have always been considered to be closely related to the Acrididae and this is borne out by their eggs. The features of tetrigid eggs that resemble those of the acridids are these: (1) a water-absorbing chorion that fragments during development; (2) a ring of micropyles near the posterior end; (3) formation of a serosal cuticle with a hydropyle at the posterior end; (4) dissolution of the white cuticle just before hatching; (5) wound-repair by the amnio-serosal fluid. 258 Hartley—The egg of Tetrix The eggs differ from those of the Acrididae in the actual structure of the chorion, except at the posterior end; in the thick secretion covering the eggs and holding them together; and in the presence of the long anterior horn. The function of the horn is not evident. There is no special air-water rela- tionship, and there is therefore no special respiratory function such as has been found in the eggs of some flies (Hinton, i960). Hancock (1902) considered that the horn plays some part in disguising the eggs when they are laid in patches of moss or lichens. He observed that when they were laid in such situations there was no attempt by the female to cover the eggs, but when laid in open ground they were always covered with soil particles. However, the female would be unable to cover them in the former situation. It has been noted that the eggs are firmly stuck together when freshly laid. The secretion usually breaks down during development under wet conditions. Under less wet conditions this might not happen and the eggs might thus be prevented from expanding laterally. It has also been noted that the horn resembles a collapsed tube in cross-section and that during development the serosal cuticle is pushed up into it. Could the horn serve as a means of allowing the egg to increase in volume without lateral expansion? There normally appears to be a doubling of volume during the development of the egg. If, however, expansion could only occur in the horn, then the egg volume could increase by an amount proportional to the length of the horn. Thus with a horn o-8 mm long the increase in volume would be 0-8/2-2 or 36%. However, there is in any case an increase in length by expansion into the horn. If this did not occur, then the diameter of the swollen eggs would be just over 1 mm. Another possibility is that lateral expansion of the egg might be limited, not by the secretion holding the eggs together, but by the hole in which the eggs have been laid. A large clump of eggs would increase its diameter by more than 1 mm over the original 2-5 to 3 mm. It seems likely that part of the function of the horn is to provide some room for the egg to increase in volume during development. The centre of the horn is hollow and some expansion of the egg occurs along it. It may also provide the exposed egg-clumps with some camouflage and provide protection against some predators. No special respiratory function can be attributed to the horn. Expansion of the egg by water uptake has been provided for in various ways by the egg envelopes. In many eggs the serosal cuticle forms a tough and elastic case which provides the structural support of the egg. The chorion may react in one of the following ways: stretching—Gryllulus (Hartley, unpublished), Staphlinidae (Lincoln, 1961), Nepidae (Hinton, 19616); stretching followed by cracking—Acrididae (Slifer, 1937; Hartley, 1961), Dytiscus (Blunk, 1914); rupture through a special area of weakness—Notostira (Johnson, 1934); expansion of a partially collapsed chorion—Tettigoniidae (Hartley, un- published). Hartley—The egg of Tetrix 259 The eggs of some of the Tettigoniidae which fall into the last category are ellipsoidal, but concave laterally, and during development they fill out. The eggs of Tetrix also fall into this category, although the chorion also stretches and cracks. The eggs of some chalcids (e.g. Encyrtidae) are bilobed, but this appears to be a method of inserting the egg through a small hole (Hinton, 1961a) and has nothing to do with increase in volume.

References BLUNK, H., 1914. 'Die Entwicklung des Dytiscus marginalis L. vom Ei bis zur Imago, i Teil. Das Embryonalleben.' Z. wiss. Zool., m, 76. HANCOCK, J. L., 1902. The Tettigidae of North America. Chicago (privately printed). HARTLEY, J. C, 1961. 'The shell of acridid eggs.' Quart. J. micr. Sci., 102, 249. HINTON, H. E., i960. 'The structure and function of the respiratory horns of the eggs of some flies.' Phil. Trans., 243, 45. 1961a. 'How some insects, especially the egg stages, avoid drowning when it rains.' Proc. S. Lond. nat. Hist. Soc. (no vol. number), 138. 1961 b. 'The structure and function of the egg shell of the Nepidae (Hemiptera).' J. insect Physiol., 7, 224. JOHNSON, C. G., 1934. 'On the eggs of Notostira erratica L. (Hemiptera, Capsidae).' Trans. Soc. Brit. Ent., 1, 1. JONES, B. M., 1958. 'Enzymatic oxidation of protein as a rate-determining step in the forma- tion of highly stable surface membranes.' Proc. roy. Soc. B, 148, 263. LINCOLN, D. C. R., 1961. 'The oxygen and water requirements of the egg of Ocypus olens Miiller (Staphylinidae, Coleoptera).' J. insect Physiol., 7, 265. ROONWAL, M. L., 1936. 'The growth changes and structure of the egg of the African migra- tory locust, Locusta migratoria migratoriodes R. and F. (Orthoptera, Acrididae).' Bull. ent. Res., 27, 1. SLIFER, E. H., 1937. 'The origin and fate of the membranes surrounding the grasshopper egg; together with some experiments on the source of the hatching enzyme.' Quart. J. micr. Sci., 79.493- 1938. 'The formation and structure of a special water-absorbing area in the membranes covering the grasshopper egg.' Ibid., 80, 437.