In Vitro Culture of Embryos in the Silkworm, Bombyx Mori L: III. Moult of Brainless Embryos in Culture

In Locustana pardalina and Locusta migratoria, it has been shown by ligaturing experiments (Jones, 1956a, b) that’secretory activity of the ventral head glands in embryos is necessary for inducing the moult and controlling subsequent events in late development’. This is an important finding which indicates that the same organized neuroglandular system as known in the larva is responsible for developmental events in the embryos of insects.

The embryonic moult is a well-known phenomenon, especially in hemimetabolous insects. But in lepidopterous insects, there was, as far as the author knows, no report of this before the work of Okada (1958) who found such a moult in Chilo suppressalis, Bombyx mori and Phassus signifer, and thought that it was a rather common phenomenon in Lepidoptera. He inclined to think that the brain-prothoracic gland system operated also in the embryo of lepidopterous insects.

While studying embryo culture in the silkworm, the author carried out investigations of the moult of decapitated embryos in this insect in vitro (instead of ligaturing experiments which could not be applied to the silkworm embryo) in order to test the possible activity of the neuroglandular system in inducing the embryonic moult.

Silkworm eggs which had been stored at 5°C. from the 50th hr. for 30 days were used as material. The pre-diapause eggs thus treated contained embryos which remained at an early stage of development as shown in Pl. 1, fig. 5, and were, as reported in the previous paper (Takami, 1958), capable of growth when they were explanted.

In these embryos the head lobe and the caudal segment were distinct at the anterior and postreior ends of the body, respectively, but there was no sign of differentiation of appendages and neuroblasts nor of the prothoracic glands, which are homologous with the ventral head glands of the locusts studied by Jones. The prothoracic glands of the silkworm first appear as a pair of invaginations in the second maxillary segment at the more advanced shortening stage of development, after the formation of appendages (Toyama,^* 1902). This point in development can be reached by the embryo at the stage shown in Pl. 1, fig. 5, after about 4 days’ incubation in the acid-treated egg.

Without acid-treatment the embryo cannot set about normal development in the intact egg, owing to the period of chilling being insufficient to eliminate diapause.

Cultures were made in almost the same way as described in the previous paper, apart from preparation of the physiological solution and culture medium. Eggs were washed in 10 % formalin for about 30 min., rinsed with sterilized distilled water, soaked in 95 % ethanol for about 1−2 min., and transferred after desiccation into the physiological solution shown in Table 1. Embryos were taken out of the eggs and their heads were cut off in this solution (Pl. 1, fig. 6). The embryo thus operated was put on a cover slip, in a hanging-drop culture over a depression slide, the medium consisting of 8 parts of the physiological solution and 1 part of yolk material from silkworm eggs (Table 2). The yolk material, addition of which was necessary for promoting development of the cultured embryos, was sucked up with a small-bore pipette from the same lot of sterilized eggs as mentioned above. The culture thus made was sealed with melted paraffin and incubated at 25°C.

Soon after the explantation, especially in the medium containing urea, a temporary enlargement, which seems to be a change intimately related with the initiation of postdiapause development, takes place in the embryos. This enlargement lasts for about half a day, and commencement of development is evident on the next day, appendages appearing on the gnathal and thoracic segments within 3 days under usual conditions of culture. Appendage formation can be used as a reliable criterion of the development of embryos, because it never occurs before the beginning of post-diapause development.

Development, which proceeded rather rapidly for the first 2 or 3 days, became slower thereafter, and detachment of the cuticle occurred in some of the decapitated embryos after the 8th day of culture (Pl. 1, fig. 7). The revolution (blastokinesis) of embryos was suppressed in culture, probably owing to the defect of the dorsal formation of cultured embryos (Takami, 1944) and to physically unfavourable conditions on the cover slip. The detachment of the cuticle first appeared at the tip of appendages where claws and bristles were just coming out (Pl. 1, figs. 4, 8, 9). This same phenomenon of moulting was observed in intact eggs which were treated with hydrochloric acid after chilling (Pl. 1, figs. 2, 3), though in these eggs it occurred at the 7th day of incubation, that is, 1 or 2 days earlier than in the cultured embryos. This delay of the moult, and the lower percentage of insects moulting in culture, must be due to a suppressed development of the embryos under unfavourable conditions in vitro ; it is not due to decapitation, because no difference could be detected between decapitated and non-decapitated embryos in culture, either in the time or in the percentage of the moult of insects moulting (Table 3).

The difference between the numbers developed and moulted in Table 3 is due to the existence of embryos in which development did not proceed to the stage of bristle formation. Though observations of these unmoulted embryos were continued for a week or more until their degeneration, the cuticle never became detached. This fact suggests that the moult does not occur before the epidermis reaches a definite stage of differentiation ; this is possibly the same in cultured and in intact embryos, that is, a stage just before bristle formation.

As the silkworm embryo cannot regenerate body segments which have been killed (Takami, 1942), the decapitated explant develops without the brain, which can be differentiated only from the head lobe. The observation that these brainless embryos can detach the cuticle is interesting, for it suggests that the’moult’ of cultured embryos does not require any hormonal action from the brain, at least for its initiation.

In these experiments the author was most concerned about the relation between the embryonic moult and the neuroglandular system, expecting that decapitated embryos would not moult in culture. But the result obtained was contrary to his expectation. The decapitated embryos moulted in the same percentage as normal embryos (Table 3). In the decapitated embryos which moulted in culture, normal differentiation of the prothoracic gland has never been observed. When decapitation was made anterior to the second maxillary segment, a few of the embryos which developed had small organs which were thought to be vestigial prothoracic glands (Pl. 1, fig. 10), but those which were decapitated posterior to the segment (Pl. 1, fig. 7) did not develop even vestigial glands. In intact eggs the prothoracic glands first appear, as invaginations in the second maxillary segment, at the shortening stage of embryos after appendage formation, and develop into the distinct band-shaped organs at the revolution stage (1 or 2 days before the occurrence of the moult), assuming the characteristic shape seen in the larva within 2 or 3 days. If the development of the prothoracic glands in the cultured embryos proceeds parallel to that in intact eggs, and if the glands have any direct relation with the occurrence of the embryonic moult, it is strange that the prothoraic glands are absent, or almost undetectable, in most of the moulted brainless embryos in culture.

The author’s results suggest that the embryonic moult occurs without any relation to the brain or even to the prothoracic glands in the silkworm, and therefore differ from the results obtained by ligaturing experiments in locusts. This difference between the two cases raises important questions about the nature of the moult in cultured silkworm embryos, which may be answered in several ways: (1) the culture medium contains some factor or factors which induce the embryonic moult; (2) the abdominal ganglia may fill the role of the brain, inducing the moult in the decapitated embryos; (3) the detachment of the cuticle in question is not a true embryonic moult such as that induced by the brain-prothoracic gland system in locusts (Jones, 1953, 1956a, b).

At present, however, we have little knowledge about the brain-prothoracic gland system controlling the events of embryonic development in the silkworm. Hattori (1959) reported on the time of differentiation of neurosecretory cells in the brain of the silkworm embryo. But it is almost impossible to find any useful data for discussing the problems in question in his brief summary. Histological investigations are now in progress to clear up the obscurities concerning the neuroglandular system in the silkworm embryo.

I am very grateful to Dr S. Shimizu, the head of the Department of Physiology, for valuable criticisms of the manuscript.