The Germinal Layers concerned in the Formation of the Alimentary Canal and Malpighian Tubules of Ephestia Kuhniella (Lepidoptera). By Mabel Drummond, Imperial College of Science and Technology. With Plates 28 .and 29. INTRODUCTION. THE origin and nature of the mid-gut epithelium of insects is of particular interest because of the differences of opinion which have existed with regard to this aspect of insect em- bryology. Eastham (1), in his review and in his work on Pier is, has shown that many of the conflicting views may be reconciled since they are due to differences of interpretation, and in some cases to faulty technique, rather than to actual variations in development. Of the recent work mention must be made of Mansour's on Calandra oryzae, Johannsen'son Diacrisia virginica, Eastham's on Pieris rapae, and Henson's on Pieris Brassicae. Mansour (4) describes the mid-gut epithelium as arising from the blind ends of the stomodaeum and proctodaeum and con- siders it to be ectodermal. This view was held by Heymons (1895) and by other workers since that time. Johannsen (3) considers the mid-gut epithelium to be endo- dermal. The endoderm rudiments first appear at the blind end of the stomodaeum and proctodaeum. Eastham (1) and Henson (2) have found that in the genus Pieris the mid-gut epithelium is endodermal, and arises from anterior and posterior rudiments which are proliferated from the germ-band independently of the stomodaeal and proctodaeal invaginations. 534 MABEL DEUMMOND Until the work of Henson, the malpighian tubules of insects, when associated with the proctodaeum, have been on account of their position regarded as ectodermal. Henson suggests that the stomodaeum and proctodaeum are not purely ectodermal. They arise on the site of the anterior and posterior rudiments which have similar positions to those occupied by the embryonic mouth and anus of Peripatus. He believes that these regions in insects and in Peripatus are homologous. In Peripatus these openings into the gut remain after the closure of the middle portion of the blastopore and are carried inwards by the developing stomodaeum and proctodaeum. Accordingly, the blind ends of the stomodaeum and proctodaeum of insects are not ectodermal 'but are com- posed of tissue homologous with the lips of the embryonic mouth and anus of Peripatus (i.e. the blastopore lips)'. In the larva two rings of cells can be distinguished which separate the ectoderm of the fore- and hind-gut from the endo- derm of the mid-gut. These rings, the interstitial rings of the canal, are not visible during embryonic development. A ring similar to the proctodaeal interstitial ring separates the main duct of the malpighian tubules from the tubules proper. Henson expresses the view that these rings are the remains of the blasto- poric areas which during development covered the tips of the stomodaeum and proctodaeum. He draws attention to the fact that the interstitial rings are in the same position as the lips of the blastopore in Peripatus. The interstitial ring of the tubules may then be due to the carrying away by the tubules of a portion of the blastopore lip during their development. He concludes, therefore, that the tubules proper are endodermal, the ring marking the end of the ectoderm. They would then be homologous with the tubules which in some insects arise from the mid-gut. MATERIAL AND METHODS. As mentioned later, I procured an abundant supply of material of the moth Ephestia kiihniella Zeller from Professor Munro. The females, which prefer to lay their eggs in the dark, were GEEMINAL LAYERS OP EPHESTIA 535 enclosed in tubes lined with black paper, and upon this the eggs were deposited. The eggs were pricked and preserved in Bouin's fixative. After fixation they were stained in borax carmine, dehydrated and cleared in mixtures of absolute alcohol and benzene. The eggs were then placed on the top of the embedding oven in a mixture of wax and benzene and left overnight, after which they were embedded in pure wax. Sections were cut 5/x. thick, and stained in Delafield's haema- toxylin. DEVELOPMENT. The early development of Ephestia closely resembles that of Pieris rapae, and only a brief description will be given. Eepeated divisions of the zygote nucleus give rise to cleavage nuclei which migrate towards the periphery and enter the cortical layer of the cytoplasm to form the blastoderm and serosa. Cer- tain of the nuclei remain in the yolk as yolk nuclei. The blasto- derm thickens to form the germ-band which, by decrease in width, comes to occupy the ventral region of the egg. Extension of the serosa takes place as a fold overlapping the blastoderm, the inner side of the fold being formed by the amnion, and this causes the germ-band to sink into the yolk. Growth in length of the germ- band now takes place by flexure of the anterior and posterior extremities of the band towards the dorsal surface of the egg. Gastrulation.—Along the median line of the germ-band with the exception of the cephalic and caudal extremities, a thickening takes place so that a middle plate becomes differ- entiated from the two lateral regions. This middle plate is invaginated into the yolk and is enclosed on the ventral surface by the overgrowth of the lateral regions. It now lies against the yolk as the body mesoderm which quickly becomes divided into segmental and inter-segmental groups of cells. During the invagination of the middle plate the evanescent endoderm cells are given off into the yolk, and there disintegrate. These cells have been noted in Pieris and Calandra. In D i a c r i s i a, however, there is no proliferation of endoderm at the time of gastrulation, but this occurs later at the time of formation of the neural groove. 586 MABEL DRUMMOND In the cephalic and caudal extremities in which mesoderm formation does not take place in the ordinary way, there are two masses of cells which are formed by budding from the germ-band at the time of invagination of the middle plate. These are the cephalic and caudal mesendoderm rudiments (fig. 1, PI. 28, and fig. 10, PI. 29). These rudiments occur in Pier is, but are not present in Calandra. In Diacrisia there are two masses of cells which are the extremities of the band of median endoderm, but which later break down. Whereas in Pier is the cephalic region of the germ-band curves through 180°, that of Ephestia curves through at least 360°, so that the extremity is directed anteriorly or even entero-dorsally. Moreover, on account of the great increase in length, the flexure is in the form of a spiral. Stomodaeum.—The stomodaeal invagination begins op- posite the cephalic mesendoderm rudiment and, according to the degree of flexure, is at first directed dorsally or, dorso- posteriorly. As the invagination deepens the cells of the rudi- ment are carried with it. The cells proliferate and those destined to form cephalic mesoderm spread towards the base, while those which will form endoderm spread over the blind end (fig. 2, PI. 29). Segmentation of the body which was indicated at the time of gastrulation has now become marked. The segments are slightly telescoped, thus allowing the embryo to straighten, so that the stomodaeum is carried through 90° and is directed anteriorly. During this time the invagination is deepening and the endoderm becomes displaced to the ventral border of the blind end (fig. 8, PL 28). It is from this endoderm rudiment and the corresponding proctodaeal one that the whole of the mid- gut epithelium is formed. In Calandra, as previously stated, the cells of the mid-gut epithelium are proliferated directly from the blind ends of the stomodaeum and proctodaeum. In Diacrisia the endoderm rudiments are differentiated later than those of Pier is and Ephestia, and first appear at the lateral angles of the stomo- daeum and proctodaeum. Oesophageal Valve.—During this time also the develop- GERMINAL LAYERS OF EPHESTIA 537 ment of the oesophageal valve begins. The valve of P i e r i s has been described by Henson. The blind end of the stomodaeum swells out and the stalk pushes into it giving it the form of a mushroom. The stalk projects into the lumen of the blind end in the form of three folds, two of which later elongate to form the valve of the larva. The oesophageal valve of Ephestia differs from that of P i e r i s in that it is formed from six tubules. While the stomo- daeal invagination is becoming directed anteriorly two groups of three tubules grow out from the blind end. Their lumina are continuous with the lumen of the blind end. The three tubules of a group arise from a common point of origin and grow back- wards over the mesoderm surrounding the stomodaeum towards the opening of the latter. The tubules respectively bear a ven- tral, right and left ventro-lateral, dorsal and right and left dorso-lateral relationship to the stomodaeum. The centre tubules of the groups are ventral and dorsal respectively (fig. 6, PI. 28). The endoderm rudiment which was on the ventral border of the blind end of the stomodaeum becomes divided into two groups of cells placed ventro-laterally and connected together by a fine strand of cells. The growth of the valve tubules pushes the endoderm rudiment backwards from the blind end of the stomodaeum. The two groups of cells remain connected together and to the tip of the stomodaeum (fig. 4, PI. 28). From each group also a fine strand of cells is being proliferated, and is extending anteriorly under cover of the mesoderm of the anterior body segments (fig.