The Hatching and Early Instars of Stenoperla prasina Newman By G. A. H. Helson, M Sc., Biological Research Laboratory, Canterbury University College, Christchurch, New Zealand. [Read before the Canterbury Philosophical Institute, July, 1934; received by the Editor. July 30, 1934; issued separately, September, 1935.] Up to the present time very little is known about the hatching of stone-flies or of the first appearance of gas in the tracheae. More is known about their eggs and manner of oviposition, but this knowledge is by no means complete. Imhof (1881) described the egg of Perla maxima and Lestage (Rousseau, 1921) described the eggs and oviposition of Perla abdominalis, but his description does not agree with that of Samal (1923). This latter author very carefully described the post-embryonic stages of Perla abdominalis, and this is the only reference to the early instars of stone-flies. Percival and Whitehead (1928) described the ova and oviposition of six different genera of stone-flies. The present paper is an account of the egg, the manner of hatching, and the early instars of S. prasina, together with a description of the first appearance of gas in the tracheae. The manner of oviposition has not been observed, but judging from the structure of the egg and the anatomy of the adult, it is probable that it is similar to that of other stone-flies. The Egg. The egg (Fig. 1) is flattened laterally to form a bilaterally symmetrical disc, which is convex on either side, is of a dark brown colour, and is perfectly smooth. Its greater diameter is 0.6 mm. and its breadth is 0.3 mm. A single female laid seventy-five eggs, each of which, when new-laid, was surrounded by a perfectly transparent, ellipsoidal envelope consisting of an albuminous substance. This envelope was three times as long and twice as wide as the greater diameter of the egg. After twenty-four hours it had disintegrated and disappeared, so that its function is presumably one of protection, protecting the egg until it has come to rest in quiet localities in the stream. Incubation. In the laboratory the period of incubation extended over ninety-one days, the last egg hatching on the ninety-fourth day. The average temperature of the water during this period was 12.8° C., and that of the laboratory 13.6° C. The minimum temperature of the water was 8.5° C., and the maximum 16.0° C., from which it is seen that the eggs can withstand a wide range of temperature. During the period of incubation the eggs were kept in a covered glass capsule, on the bottom of which there was sand. A water-weed (Elodea) was growing in the water, fresh quantities of which were added from time to time.

Hatching. As the time for eclosion draws near the eggs increase in thickness by 0.05 mm., and increase slightly in convexity. At this stage the chorion appears to be distended by an internal pressure. It is not possible to deform it with a needle; the eggs rupture when this is attempted. The larvule lies on its side coiled round the greater perimeter of the egg with the head and tail meeting. The cerci extend externally over the thorax, and the antennae lie externally over the abdomen, while the legs are folded together and occupy the central portion of the egg. Increased activity of the larvule marks the commencement of hatching, and eclosion is affected by the alternate violent straightening and contracting of the body. At each flexion the head and abdomen oppose one another and exert a pressure on the chorion. Major flexions occur every seventeen seconds, whilst smaller ones occur irregularly in the intervals between. After three or four violent flexions a slit round the greater perimeter appears in the chorion between the two halves of the egg. As flexing proceeds this slit enlarges until of sufficient size to allow the abdomen to be ecloded; the larvule then wriggles violently till the two halves of the chorion separate and hatching is completed. After this the newly emerged larvule is exhausted and lies quiet for about half an hour, when it once more resumes activity. The total time taken to hatch is twenty minutes. The First Appearance of Gas in the Tracheae. Gas first appears in the tracheae of the head almost immediately after the eclosion of the abdomen, and by the end of the seventh second has passed down the main lateral trunk as far as the thorax. It continues to progress backwards, and two seconds later the tracheae of the first abdominal gills are filled. Gills two and three are filled two seconds after the first. After this the gas extends along the lateral, abdominal trunks more slowly, taking five minutes to reach the base of the cercus, another five minutes elapsing before it reaches the end of this latter. The total time taken to fill all the main tracheae with gas was ten minutes eleven seconds. In the light of Wigglesworth's recent research (1931) on the hatching of insects, the rapidity with which the gas extends along the lateral trunks is understandable. The increased activity of the hatching larvule probably causes an increase in the osmotic pressure of the tissue-fluids, by which the fluid in the tracheae is absorbed into the tissues. Where the gas which replaces this fluid comes from, or what its composition is, is a matter for conjecture, since its first appearance is in the head and not in the gills. It is possible, however, that it may result from the products of metabolism during the period of increased activity, being replaced later with oxygen by diffusion from the surrounding water during the period of quiescence. The First Instar. The first instar (Fig. 2) resembles the full-grown nymph described elsewhere by the present author (1934).

In the head the chief difference is the absence of compound eyes, three ocelli occupying the position in which these later appear. The antenna is only eleven-jointed instead of being multiarticulate; the mouth-parts, however, are of the typical biting type, and resemble those of later instars. The thorax and legs present no notable variations, except that the latter lack the tarsal adaptations of the older nymph. The only difference exhibited by the abdomen is the presence of the three segmented, tracheal gills on segments three, four, and five, instead of five on segments one to five inclusive. These gills are very long, and are about half as long as the abdomen. The cercus is five-jointed. The Tracheal System. The tracheal system conforms to the same general arrangement of the nymph, namely, two longitudinal lateral trunks extending from the head to the cercus. The typical Y-shaped tracheae to the legs are present as are also tracheae to the gills from the lateral trunks. The Alimentary System. The alimentary canal shows no marked deviation from that of later instars. The oesophagus extends to the pro-thorax, the crop and gizzard to the first abdominal segment, and the mesenteron to the sixth segment. The oesophageal valve is well developed, even at this stage, and definitely acts as a valve between the gizzard and the mesenteron. Peristaltic waves pass along the fore and hind gut, but whether these are synchronous or not is not known. The Second Instar. Since the larvules hatched in captivity could not be kept alive for more than a week, it was found necessary to determine the instars of early material collected in the field by mathematical deduction. The instar depicted in Fig. 3 was deduced to be a second instar by this means. Dyar's law (Imms, 1925) states that the width of the head increases 1.44 at each ecdysis, and using this constant it was found that the head of the nymph in Fig. 3, which will be designated B, was approximately 1.44 times the width of the head of the nymph represented in Fig. 3, which will be called A. Again Przibram (1931) has found that the growth rates of various parts of the body bear a definite ratio to each other after each ecdysis, and while his constant of 1.26 for the growth of the tibia was found to be inapplicable in S. prasina, this constant does apply to the ratio between the total length of the head from clypeus to cervicum and to the ratio between the total lengths of the body after each ecdysis. Thus the head of B is 1.26 times the length of that of A, and in the same way the total length of B is 1.26 times the length of A. Williams (Przibram, 1931), describing the life history of an Indian mantid, Gongylus gongyloides, made the remark in 1904 that at each moult it increased its total length by one-fifth. Now, adding one-fifth part to the whole part present at the previous moult, the quotient 1.25 is arrived at. Numerous other examples tend to support the accuracy of this quotient. From the above mathematical data it is deduced that B represents a second instar.

This instar differs only in detail from the first. There is an increase in size and, from Przibram's generalisation, a corresponding doubling in weight. The antennal segments have increased to fourteen and those of the cercus to seven. The mouth-parts are the same and the compound eyes are still absent, whilst the epicranial suture has become evident for the first time. At this stage the tarsal adaptations of the older nymphs appear. The chief difference, however, is the appearance of gill rudiments (r.g.) on segments one and two, together with the tracheae supplying them. The other three gills are still relatively large, but show very little increase in length. The body is perfectly transparent, and the green of the gut contents shows through very clearly. Feeding. The food of the early instars of S. Prasina consists of diatoms and algae, as in other genera of stone-flies. The particular diatom fed upon by a nymph taken from the Temuka River was a species of Navicula, together with filaments of algae. Of the first instars hatched in captivity only one endeavoured to feed when placed in a culture of Protozoa, Rotifers, Tardigrada, Algae, Elodea, and Nitella. This individual ate three Rotifers, which caused it great discomfort; it died shortly afterwards. The milling action of the gizzard could be clearly seen. At each peristalsis a vigorous contraction occurred in the gizzard, compressing the food until it was masticated; then, and not before, was it allowed to pass into the mesenteron. Evidently the enzymes present at this stage were not capable of acting upon a carnivorous diet, for the Rotifers in question passed unaltered right through to the rectum, in which region they were lodged when the insect died. In spite of the change from a herbivorous diet in these early instars to a carnivorous one in later, the mouth-parts are of the typical biting type throughout. Bibliography. Helson, G. A. H., 1934. Bionomics and Anatomy of Stenoperla prasina, Trans. Roy. Soc. N.Z., vol. 64, pp. 214–248. Imhoff, L., 1881. Beitr. zur Anat, von Perla maxima, Inaug. Diss. Aara. Imms A. D., 1925. A Textbook of Entomology, first edition. Methuen and Co., Ltd., London. Percival. E., and Whitehead, H., 1928. Observations on the Ova and Oviposition of Certain Ephemeroptera and Plecoptera, Proc. Leeds Phil. Soc., vol. 1, pp, 271–288. Przibram, H., 1931. Connecting Laws in Animal Morphology, Univ. Lond. Press, London. Rosseau, E., Lestage, J., and Schouteden, H., 1921. Les Larves et Nymphes Aquatiques des Insectes d'Europe, vol. 1, Office de Publicite, Brussels. Samal. J., 1923. Etudes morph. et biol. de Perla abdominalis, Ann. de Biol. Lac. 12. Wigglesworth, V. B., and Sikes, E. K., 1931. The Hatching of Insects from the Egg and the First Appearance of Air in the Tracheal System, Q.J.M.S., vol. 74, pp. 165–192.

Fig. 1.—The egg of Stenoperla prasina (side view). X 80. Fig. 2.—The first instar of S. prasina, showing the three abdominal gills. The gut and tracheae show clearly through the transparent cuticle.

Fig. 3.—The second instar of S. prasina, showing the rudiments of gills on segments one and two. r.g.: rudiment of gill.