1. The use of ultrasonics as a tool for the experimental study of embryonic processes in Drosophila has been investigated.

  2. It has been found that developmental changes can be induced by the application of ultrasonic waves at intensities sufficiently low to obviate the possibility of cavitation damage or chemical change.

  3. The effects of ultrasonic waves of various intensities on different developmental stages has been studied by direct visual observation during treatment and in permanent preparations of sectioned material at subsequent developmental stages.

  4. Treatment at preblastodermal stages was usually followed by readjustment and normal but delayed development; in other cases death ensued without further development.

  5. Treatment of eggs in late cleavage stages with ultrasonic intensities of 0·5 to 1·2 watts/cm.2 for 30 seconds produced abnormalities of spindle, displaced centrioles, and induced polyploidy.

  6. Treatment at the syncytial blastoderm stage, with ultrasonic intensities 0·3 to 0·5 watts/cm.2 for 30 seconds, resulted in the production of a high proportion of developmental abnormalities which were classified into five different categories: (a) death without further development; (b) proliferation of cells without differentiation; (c) differentiation without organization; (d) abnormal organization; and (e) slight abnormalities in organization.

  7. The possible causes of the different types of abnormalities in embryonic development after treatment with ultrasonics are considered, and are discussed in relation to abnormalities in embryogenesis which have been produced in Dipteran embryos by other means, and in the light of the general morphogenetic situation existing in Diptera and certain other insect orders. Further applications of the method to developmental studies are suggested.

The work of Seidel and his school (reviewed Seidel, 1935, 1936) showed that embryonic development within the class Insecta ranges from the indeterminate or regulative type in the Odonata to completely determinate or mosaic development in the Diptera. Between these two extremes are insects in which the developmental fate of the egg is not determined at the time of fertilization, but which becomes determined at varying times afterwards. Actually the scale is a relative one, for development eventually becomes completely determined in the Odonata, and there is experimental evidence (Howland & Sonnenblick, 1936) which suggests there is some regulative power in the Dipteran egg prior to fusion of the gamete nuclei.

Experimental methods used to such great advantage in studies of vertebrate development cannot be used in studying the insect egg, as the turgor pressure and fluidity of the egg contents make transplantation studies and the culturing of embryonic tissues in vitro impossible in most forms. The many alternative techniques which have been devised for studying embryological processes in insects have been reviewed by Richards & Millar (1937) and Krause (1939).

Many workers have studied the effects of treatment applied to the egg in later post-embryonic developmental stages; for example Howland & Child (1935) and Howland & Sonnenblick (1936) studied the effects of embryonic punctures on development of structures in the adult, and Geigy (1931a) the effects of ultraviolet irradiation of the egg on imaginal structures. Waddington (1942) has pointed out the dangers of using defect experiments as a means of discovering developmental potentials.

Experimental methods which have been used in purely embryological studies on the Diptera include:

  1. Cauterization (Reith, 1925—Musca; Strasburger, 1934—Calliphora; Howland & Robertson, 1934—Drosophila).

  2. Irradiation with ultraviolet light (Geigy, 1931bDrosophila; Aboim, 1945—Drosophila).

  3. Constriction (Pauli, 1927—Calliphora and Musca; Rostand, 1927—Calliphora).

  4. Centrifugation (Pauli, 1927—Calliphora and Musca; Howland, 1941— Drosophila).

  5. Treatment with X-rays (Sonnenblick, 1940—Drosophila; Sonnenblick & Henshaw, 1941—Drosophila).

Although these methods have given us much valuable information about embryonic development in Diptera (especially the experiments of Reith and Pauli which demonstrated the mosaic nature of development, the totipotency of cleavage nuclei, and the importance of the periplasm in determining the fate of nuclei which enter various regions of it) they are not completely satisfactory tools. Microcautery and radiation treatments result in the destruction of regions of the egg; the chromosomes are also affected by the use of ionizing radiations. Constriction experiments are tedious and difficult because of the size of the egg, and are infrequently successful. Centrifugation experiments require treatment of the developing eggs for long periods of time even at high speeds to shift the periplasm.

The use of ultrasonic waves as an experimental method in insect embryology has not yet been fully exploited. Ultrasonic treatment may cause a ‘stirring round’ in certain regions of the egg but no nuclei need be destroyed. Fritz-Niggli (1950,1951) has studied the effects of ultrasonics on development in Drosophila, but although all developmental stages were treated, her histological studies were confined to prepupal, pupal, and adult stages. A review of biological studies with ultrasonics is given by Dognon, Biancani, E., & Biancani, H. (1937), and an account of the effects of ultrasonics on mitosis by Selman (1952). The apparatus used in these experiments was similar to that developed by Selman & Wilkins (1949) for the treatment of biological material under controlled conditions. It was found that when the early developmental stages of Drosophila embryos were treated for short periods with ultrasonic waves at intensities low enough to obviate the danger of cavitation and chemical damage, the subsequent embryonic development was abnormal. A preliminary report of these experiments has already been published (Selman & Counce, 1953).

Eggs were collected at half-hour intervals from rapidly laying Oregon K flies of stock maintained in this laboratory, and were transferred to the central region of thin agar disks for storage in Petri dishes under moist conditions at 25° C. Immediately before treatment the agar disks were placed in the treatment vessel so that the eggs faced the ultrasonic generator (Text-fig. 1). The ultrasonic waves at a frequency of 1 Mc./sec. were applied through a water medium maintained at room temperature, using a uniform beam of ultrasonics of known intensity. The ultrasonic intensities were known from a calibration chart for the generator, previously prepared from measurements made similarly to those described by Selman & Wilkins (1949). After treatment the eggs were replaced in the Petri dishes for further incubation at 25° C. until they were needed for fixation.

A 6:16:1 formol : alcohol : acetic acid mixture was used as fixative (Darlington & La Cour, 1942, after Smith). To ensure rapid and even fixation the vitelline membrane was pierced with the tip of an exceedingly fine tungsten needle as soon as the egg was placed in the fixative. After fixation for 2 to 24 hours, eggs were dehydrated through a butyl alcohol series (Darlington & La Cour, 1942) containing eosin, infiltrated with 45 ° C. paraffin with an equal amount of phenol, changed after 12 to 16 hours into fresh 45° C. paraffin and embedded in hard wax. Serial sections were cut at 4 microns and stained in Heidenhain’s iron haematoxylin according to the schedule suggested by Sonnenblick (1950).

None of the eggs treated in the main experiment were dechorionated. The optically opaque chorion was transparent to ultrasonics and did not hinder fixation, while the chorionic filaments were useful guides to the orientation of abnormal embryos. In subsidiary experiments, however, dechorionated eggs were treated with ultrasonics using a special small generator mounted on a microscope stage (cf. Harvey, E. N., Harvey, E. B., & Loomis, 1928) and this allowed direct visual microscopic examination of the living egg to be made by transmitted light while the ultrasonic treatment was actually being given. In this way disturbances caused by the treatment could be seen. With this apparatus, however, it was not possible to determine precisely the intensity of the ultrasonic waves which were applied, nor was there any temperature control, and for these reasons the microscope stage generator was not used in the main embryological or cytological studies.

Preliminary experiments showed that treatment at any particular ultrasonic intensity for a given time produced a maximum lethality when the eggs were at the stage of migration of the nuclei to the periphery and the formation of the syncytial blastoderm (Text-fig. 2). This was the case when the lethality of the treatment was reckoned by counting the numbers of eggs which hatched or failed to hatch, and also from estimates based on the subsequent numbers of flies.

In the main experiment the following early developmental stages were treated with ultrasonics: maturation or early cleavage (immediately after collection); late cleavage stage (1 ± hr. after deposition). Several ultrasonic intensities were used: 0 ·05, 0 ·1, 0 ·3, 0 ·5, and 1·2 watts/cm.2; all treatments were of 30 seconds duration at room temperature. Many eggs were treated simultaneously so that eggs from the same batch could be fixed at a number of times after treatment.

Table 1 shows the numbers of eggs treated at each developmental stage and at each ultrasonic intensity, together with a classification of the subsequent conditions of development reached by the embryos at the time of fixation. Table 1 was prepared from microscopic examination of permanent stained serial sections.

Table 1.

Number and classification of eggs fixed after treatment at different ultrasonic intensities

Number and classification of eggs fixed after treatment at different ultrasonic intensities
Number and classification of eggs fixed after treatment at different ultrasonic intensities

Ultrasonic intensities lower than 0·1 watt/cm.2 had little or no effect on embryonic development irrespective of the stage at the time of treatment.

Treatment of preblastoderm stages

When ultrasonic waves of intensity about 0 ·5 watts/cm.2 were applied during cleavage stage to eggs mounted on the microscope stage generator, a slow rotary movement of a large proportion of the central region of the egg was observed; the yolk granules scattered sufficient light for these movements to be followed. The periplasm was more resistant to displacement, for its position remained unaltered. If the treatment were stopped temporarily and then restarted again, a higher intensity was required to set the central region in motion once more.

Eggs treated during preblastodermal stages at intensities 0·3, 0·5, and 1·2 watts/cm.2 and fixed immediately showed clearly the outline of the disturbed regions (Plate 1, fig. 1). The staining reactions of the two regions of cytoplasm differed, those parts which had been moved staining more lightly than adjacent regions; the disturbed parts were also without yolk granules at their periphery. The cytoplasm of the egg was more vacuolar than normal.

Treatment prior to blastoderm formation usually had one of two results: either the ooplasmic contents were so disordered that reconstitution did not take place and the egg subsequently degenerated without further development, or reconstitution occurred and development was normal but slightly delayed (see Table 1). All eggs treated at this stage at an intensity of 1·2 watts/cm.2 failed to show any sign of organization subsequently. They exhibited abnormal cytology (see later section) which may have caused death, but degeneration set in before any sign of blastoderm formation could be seen. The one embryo treated at this intensity in which abnormal organogenesis was observed (Table 1) was in all probability at a somewhat later stage in development than the other eggs treated at this time. Eggs collected from rapidly laying females are usually at the same stage of development at the time of deposition (pre-fusion of the gamete nuclei), but occasionally eggs may be retained in the uterus of the female for varying periods of time after fertilization. The development of such eggs continues in the uterus, and they may even be deposited just before the emergence of the larva.

After treatment at lower intensities the slight abnormalities observed in embryos in which organogenesis had been completed were usually related to abnormal distribution of yolk granules. These were found in the proventriculus, although they do not normally occur there; in other instances abnormally large quantities of yolk were included in the nervous system. Some abnormalities in external segmentation were also observed. It is not certain whether those embryos showing slight abnormalities might not have developed into adult flies. It is possible that embryos of this sort account in part for the delayed mortality observed by Fritz-Niggli (1950, 1951), which was confirmed by us during the course of this work. Such embryos may emerge from the embryonic membranes only to die at a later stage in development, but there is no proof of this.

Treatment of syncytial blastoderm stages

Using the ultrasonic microscope-stage generator it was observed that the pole cells were the part most easily set in motion when embryos were treated at the syncytial blastoderm stage. Intensities of 0·3 to 0 ·5 watts/cm.2 caused them to spin rapidly and the disturbance frequently caused a break in the posterior region of the blastoderm through which yolk and cytoplasm from within the interior of the embryo could be seen to flow. There was also a slow progression of blastoderm nuclei and cytoplasm towards the area of disturbance. In a few cases small air-bubbles in the water on the outer surface of the embryo were caused to vibrate in the ultrasonic beam and gave rise to secondary disturbances. Those breaks which occurred in the syncytial blastoderm, not at the posterior end, were probably produced in this way.

Treatment at the syncytial blastoderm stage was found to give rise to the greatest proportion of embryos showing abnormal development. At this stage ultrasonic intensities of 0 ·3 to 0·5 watts/cm.2 were found to be the most effective in producing abnormalities. Accordingly a large number of embryos were fixed at several intervals after treatment at this intensity applied to eggs 2 hours after deposition; 80 per cent, of these embryos showed some kind of abnormal development. The abnormalities found in the serial sections are described here in developmental sequence.

(a) Syncytial blastoderm stage (embryos fixed immediately after treatment)

In some embryos a vortex-like disturbance of the pole-cells alone was to be seen. More usually in the region of the disturbance there was a mixture of cytoplasm, yolk granules, pole-cell nuclei, and blastoderm nuclei (Plate 1, fig. 2). Frequently an irregular darkly stained mass was found in the abnormal region, probably as a result of fragmentation of yolk granules (Plate 2, fig. 9). The size of the abnormal region varied; this may have been due to changes in the mechanical strength of the blastoderm itself, which might increase with the number of nuclei included in it as the period of cell-wall formation approached.

When subsidiary disturbances had occurred in other regions of the egg the break in the blastoderm was accompanied by the movement of cytoplasm, yolk, and blastoderm nuclei to the surface of the embryo. With very small breaks in the blastoderm, only yolk granules moved to the egg surface. In a few embryos clefts in the cytoplasm perpendicular to the egg surface were formed (Plate 1, fig. 3). Nuclei lined up along these clefts which probably occurred as ruptures produced as a result of strain.

(b) Cellular blastoderm, gastrulation, and histogenesis

The abnormalities which occurred in gastrulation depended upon the extent and position of the original disturbed area. In undamaged regions the cells which formed were typical elongate blastoderm cells. In those embryos in which the damaged region was large no gastrulation movement of any kind occurred, although cellular proliferation and rarely some differentiation of cell types took place. Some embryos (e.g. Plate 1, fig. 4) gave indications of gastrular differentiation, such as the formation of the germ-band in undisturbed areas; later, invagination of the anterior midgut rudiment took place.

In other embryos at this stage more complete gastrulation movements occurred, but various abnormalities in the processes of gastrulation could be observed. The cephalic furrow did not form in some; in others it formed but was incomplete, or the orientation of the cleft was abnormal. In yet other embryos the cleft remained a conspicuous feature long after it ceased to be obvious in normal embryos of the same age. Abnormalities were also observed in the formation of the posterior midgut invagination. In those embryos in which the damage at the posterior pole was considerable, the posterior invagination did not form. The invagination formed in other embryos, but the elongation took place from the posterior pole or the ventral surface. In some embryos the posterior invagination formed and moved anteriorly but the pole-cells were not included within it; when this happened the shape of the posterior invagination was affected, because the walls of the invagination were apposed throughout their length instead of assuming a sack-like shape. The germ-band was asymmetrical in those embryos for which the damaged region extended farther along one side than the other.

Later, the anterior midgut did not form in some cases or it formed on one side only. Formation of the stomodaeum was frequently affected; it usually failed to invaginate. In other embryos it formed but was abnormal in position. The presence of free yolk on the surface caused the shape of the embryo to be distorted.

By 6 hours, nuclei which were in or bordering on the abnormal region had become very large (Plate 1, fig. 4) and showed some clumping of chromatin. It is probable that these nuclei increased in size by endomitosis as no mitotic figures were found. The pole-cells remaining in abnormal positions retained, in some instances, their distinctive cytological characteristics for several hours.

In some embryos the only differentiation which was obvious was the formation of the primitive gut; in others there were present small cells similar to ganglion cells. Mitotic activity continued for some time in even the most abnormal embryos.

During histogenesis many of the abnormalities which became conspicuous later in development could already be discerned. Particularly obvious at this time were abnormalities in hypodermal differentiation and in the development of organs which originate from the superficial ectoderm.

Abnormalities were frequently observed in the orientation of ganglion cells and the neuroblast cells from which they were formed. Whereas in normal embryos the unequal division of the neuroblast takes place in a direction perpendicular to the ventral surface with the small cell which is to become a ganglion cell budding off at the end nearest the centre of the egg (Poulson, 1950), in treated embryos the neuroblast divisions may be in a plane parallel to the ventral surface and the relative positions of ganglion cell and neuroblast cell may vary.

(c) Organogenesis (12 to 20 hours)

The embryos fixed in this group revealed the end results of treatment. The expression of abnormality varied, depending upon the position and size of the regions affected at the early stage. The abnormal embryos were classified into five groups.

  • The first group of seven embryos showed little or no development after treatment. Six of these showed mitotic divisions and in a few instances the spindles appeared abnormal, although subsequent degeneration generally made detailed cytological examination impossible. In one embryo the nuclei were very large with clumped chromatin, but these were arranged round the periphery as are normal nuclei at the syncytial blastoderm stage. The abnormal nuclei resembled those described previously as having developed in regions directly affected by the treatment.

  • The second group of eleven embryos showed cellular proliferation without differentiation into the various types of tissue characteristic of the embryo (Plate 1, fig. 5). The disturbed region was always very large in these embryos.

  • In another group of twenty embryos differentiation but no morphological organization had taken place. The shape of these embryos was usually reminiscent of gastrular form, but in some (Plate 1, fig. 6) the resemblance was obscured by the extent of the damaged regions. Differentiation of the nervous system into ganglion cells and fibres always occurred in these embryos although the fibres were not well organized and might be only slightly in evidence. Mesoderm was present and in some cases had differentiated into elongate cells; elongation of cells, however, was never followed by fusion, and no muscles were formed. The gut was represented in some cases but not in others; often the cells were clumped and disorganized, but tubular portions sometimes appeared, usually in the anterior end. These embryos were no doubt those in which early gastrulation processes had been very abnormal.

  • Twenty-seven embryos showed markedly abnormal organization, which was expressed in several different ways. In some embryos spatial relationships between organs were distorted; in some, certain organs and tissues showed abnormal differentiation; in others certain organs and tissues were incomplete or lacking. Various combinations of these abnormalities were also found (Plate 1, figs. 7–8; Plate 2, figs. 9–12).

    A common abnormality was the differentiation of the posterior spiracles and the posterior spiracular atrium at the dorsal surface near the middle of the embryos, with the anus also in the dorsal midline (Plate 1, figs. 7 and 8). In one embryo (Plate 2, figs. 9 and 10) the posterior spiracles developed in the posterior region, but with the opening of the spiracles deep in the interior of the embryo. The posterior end of this embryo resembled a hollow ball one side of which had been pushed in by pressure applied with the thumbs. In this embryo the anus opened at the posterior end.

    Abnormalities were frequently found in the formation of the pro-ventriculus as a result of disturbed spatial relationships between the oesophageal and gastric portions of the structure. The development of the two portions seems quite independent, for in embryos in which the oesophageal component was lacking, or was not in contact with the gastric portion, the gastric portion still differentiated and the peritrophic membrane was sometimes secreted.

    Various abnormalities in the spatial relationships of the salivary glands have been noted. In one embryo (Plate 2, fig. 11) the glands were on the same side of the embryo; in another the distal end of one of the glands was directed towards the anterior rather than the posterior end as in normal embryos. The presence in the glands of a staining substance indicated that the secretory function of the glands was not impaired.

    Involution of the head rarely took place, and the shape and position of the cephalopharyngeal apparatus and mouth hooks were abnormal in the three embryos in which there had been some involution (Plate 1, fig. 8, and Plate 2, fig. 9).

    The most spectacular of the abnormal spatial relationships was the formation in two embryos of the gonads at the anterior end near the region of the mouth parts (Plate 2, fig. 12).

    Differentiation of organs and tissues was abnormal in many embryos. In only one of the embryos examined did normal gut differentiation occur; differentiation of the visceral musculature was also rudimentary. Clumping of muscle-cells was frequently observed. In one embryo (Plate 2, fig. 11) no organs or tissues derived from the mesoderm were formed, although undifferentiated mesoderm cells were present.

    Some incomplete formation or absence of organs and tissues occurred in every embryo showing abnormal organogenesis. Some cases, such as the failure of the tracheal branches to unite, were purely mechanical in nature. Mechanical abnormalities also account for the absence of the posterior gut rudiment, foregut and stomodaeum, as well as absence of gonads. In almost half of the embryos in which organization was aberrant, the salivary glands were lacking; this was related to abnormalities in the differentiation of the superficial ectoderm.

    The hypodermis was incomplete in all treated embryos and was totally lacking in one (Plate 2, fig. 12). Those portions of the embryo which develop from the frontal sac (which moves superficial ectoderm from the surface to the interior by invagination) were lacking in all but three embryos. In all embryos showing abnormal organogenesis, mesoderm and nervous tissue were present together with at least some portions of gut, although the latter might occasionally consist only of clumps of cells.

    Segmentation was abnormal in all these embryos. This was in part due to the absence of hypodermis and the abnormal differentiation of muscles, but free yolk at the surface also resulted in exaggerated segmentation (Plate 1, fig. 7).

  • A group of thirteen embryos showed slight abnormalities, the most frequent of which was the absence of gonads or slight abnormalities in segmentation. In a few cases there was abnormal distribution of yolk granules, either in tissues or in regions of the gut where yolk is not normally found. In one embryo some muscle-cells, which had undergone elongation and partial fusion, were completely enclosed by nervous system.

  • Twenty-seven of the treated embryos which were fixed at this age were apparently normal in all respects.

Cytological Abnormalities

Although some cytological abnormalities were produced by treatment at the syncytial blastoderm stage with ultrasonics of intensity 0 ·3 to 0·5 watts/cm.2 (see above), it was found during ancilliary experiments that cytological abnormalities were produced most frequently after treatment at a slightly earlier stage with ultrasonics of higher intensity. The normal cytology of the material is described Forty-nine eggs were fixed between 5 and 15 minutes after treatment 1 hour after deposition using ultrasonic intensities between 05 and 1·2 watts/cm.2 for 30 seconds; the permanent serial sections subsequently prepared were examined using an oil-immersion objective.

Nine eggs showed cleavage divisions with abnormalities of spindle and centriole which were probably caused by the streaming motion of cytoplasm and yolk during treatment. Thus instances were found of centrioles displaced from their interphase nuclei. One centriole was situated equidistant from two nuclei in late prophase; it formed the pole of two mono-polar spindles which were forming one in connexion with each set of chromosomes, there being no other centriole associated with either nucleus. A dipolar spindle with centrioles was found outside and slightly displaced from a nucleus whose nuclear membrane was intact (Plate 2, fig. 14). One end of a spindle at metaphase was found to be split into two parts each of which had a single centriole at a separate pole, while the opposite half of the spindle was normal: the spindle therefore appeared tripolar. Another metaphase spindle was found split in a direction perpendicular to the metaphase plate (Plate 2, fig. 15).

There were several instances of chromosomes displaced laterally at metaphase without, however, being completely separated from the spindle. In two eggs no chromatin at all could be found in the many cleavage divisions, which were recognized by the presence of apparently normal, but empty, spindles together with their centrioles and asters (Plate 2, fig. 16). The cytoplasm of these eggs had been disturbed by the treatment to the usual extent but appeared otherwise normal, with yolk granules intact; moreover the staining was unimpaired, as could be gauged from the deeply stained appearance of the condensed chromosomes of the polar bodies. It was most striking to note how in the majority of cases the spindles and asters of the mitotic figure protected the cleavage divisions from mechanical disturbance by streams of moving yolk and cytoplasm during treatment. Twenty eggs from this group showed cleavage divisions which were apparently quite normal in every respect, in spite of the fact that the contents of the egg had obviously been stirred round. Two eggs showed only normal mitotic figures, but these exhibited a definite loss of synchrony not attributable to a fixation gradient. Four eggs of the group were normal but of a much later stage of development, while thirteen others were too badly damaged to analyse.

Eight eggs were fixed 3 to 4 hours after ultrasonic treatment at intensities 0 ·5 to 1 ·2 watts/cm.2 applied during the late cleavage stage (i.e. 1 hour after deposition). One of these eggs was an abnormal gastrula, but the other seven showed nuclear proliferation with no sign of nuclear migration to form a syncytial blastoderm. Each of these seven eggs contained obviously polyploid nuclei and the polyploidy must be presumed to have developed subsequently to and as a result of the treatment. Large numbers of metaphase figures with polyploid chromosome sets were found (e.g. Plate 2, figs. 18 and 19); some of these had apparently normal spindles, centrioles, and asters, while in others the spindles and asters were irregular and of indefinite polarity, and the centrioles were sometimes lacking. Groups of centrioles, each with its aster, were to be seen in the cytoplasm remote from any nearby nuclei. Enormous resting-stage nuclei of irregular shape were also present (Plate 2, fig. 13); one of these was 40μ. long. One of these eggs was found to contain 8 diploid metaphase figures (including one with a split spindle), 9 polyploid metaphase figures with normal spindles and centrioles, and about 80 polyploid metaphase figures either with centrioles absent or with the spindle abnormal. There were also 3 normal diploid resting-stage nuclei in the same egg together with about 120 abnormally large resting-stage nuclei of a wide range of size and shape. In the cytoplasm about 190 free centrioles with asters were found.

In many eggs which were treated with ultrasonics at preblastodermal stages, reconstitution of the ooplasmic contents followed and development continued normally. Pauli (1927) observed a similar reconstitution after centrifugation of Calliphora eggs before blastoderm formation; the stratification of the ooplasmic contents by centrifugation, however, is not similar to the redistribution of the egg contents as a result of ultrasonic treatment. Partial reorganization was never observed in eggs which had been subjected to ultrasonic treatment at this stage; either no reorganization followed and degeneration began, or embryos developed which were normal except for very slight abnormalities in a few cases.

It is possible that interference with mitosis is responsible for some of the early deaths in eggs treated with ultrasonics at preblastodermal stages. Howland (1941) frequently obtained abnormalities of spindle configuration and function after centrifuging early egg stages of Drosophila, and these were probably related in part to changes in the nature of the egg cytoplasm. Some evidence has been presented for similar cytoplasmic changes taking place in some eggs on treatment with ultrasonics, and it is possible that these caused some of the abnormalities in spindle function and led to the observed polyploidy. The displacement of centrioles and some cases of distortion of spindles in our experiments are, however, more readily attributable to direct mechanical effects caused by streams of heavy yolk granules moved by the action of the ultrasonic waves. It may be significant that the cytological abnormalities which Bessler (1952) obtained in gastrulae of newt by ultrasonic treatment were obtained in a yolky material. In Drosophila eggs the absence of cell membranes between the cleavage nuclei before blastoderm formation makes the streaming movements caused by the ultrasonic treatment all the more effective in producing cytological abnormalities; from this point of view therefore the material is more favourable than the meristematic tissues used by Selman (1952) for studying the cytological effects of ultrasonics. However, the chromosomes of the present material are not particularly favourable for the observation of induced abnormalities.

It is probably coincidence that the period of maximum lethality for ultrasonic treatment and various other treatments such as X-radiation (Packard, 1926, 1935) and alpha-radiation (Hanson & Heys, 1933) is the syncytial blastoderm stage. It is not surprising that the nuclei are highly susceptible to the effects of radiation at this stage for they are undergoing rapid division near the outer surface of the egg. On the other hand, the lethal effects of ultrasonic treatment at this time seem only rarely to be related to nuclear abnormalities; more frequently they are caused by changes in the blastoderm which occur as a result of the rotation of the pole-cells, the formation of the vortex, and the concomitant displacement of the egg constituents.

None of the experimental methods which have been applied to insect eggs are comparable in their effects with those obtained with ultrasonic treatment. Centrifugation, which like ultrasonic treatment can result in a shift in the contents of the egg at early stages, is not able to cause a shift in the blastoderm once it has been established (Pauli, 1927; Howland, 1941). The other methods are dependent upon the removal of restricted regions of the egg, without altering the positions of nuclei. In eggs subjected to ultrasonic treatment, as a result of the progression of the blastodermal cells towards the region of disturbance, the spatial relationships within the blastoderm are altered. In addition to the change in position of nuclei which are supposedly ‘determined’ when they enter the peripheral cytoplasm, there is in all probability a movement of the innermost regions of the cytoplasm quite independent of nuclear movements, so that the cytoplasmic environment of nuclei may be substantially different to that in normal embryos. The abnormalities in gastrulation which occurred after treatment are easily explained by considering the mechanical difficulties arising as a result of the presence in the embryo of abnormal regions caused by disturbance at the time of treatment. Some abnormalities are the result of changes in position of blastodermal nuclei, so that the cells which formed the stomodaeum, for instance, were more ventral than in normal embryos. Failure of shortening during the middle period of embryogenesis sometimes caused the posterior tracheal regions and the anus to occupy very abnormal positions. Other morphogenetic movements occurring at the same stage, such as head involution and dorsal closure, were also affected.

The end results of treatment are of more interest. In some embryos considerable proliferation of cells took place after treatment, although there was no cellular differentiation. Pauli (1927) has described embryos which developed from eggs which had been centrifuged and in which there was multiplication of cells without differentiation; these all developed from eggs which had been centrifuged for long periods of time with the result that the ooplasmic contents became very disorganized. Sonnenblick (1940) and Sonnenblick & Henshaw (1941) found many embryos showing this phenomenon after one parent had been treated with X-rays; frequently part of the cytoplasm in these embryos remained non-cellular but stained deeply with basic dyes (cf. Plate 1, fig. 5). In the cases described by Pauli cytoplasmic factors were probably involved, and it is probable that the underlying cause of the abnormality produced by ultiasonic treatments is similar. It is not impossible, however, that chromosomal factors may have caused the abnormality, as they most probably did in dominant lethals which were induced with X-rays by Sonnenblick (1940).

Differentiation of tissues without any organogenesis in embryos after ultrasonic treatment at the syncytial blastoderm stage seemed to be related to the failure of normal gastrulation. It would seem that even in mosaic eggs certain morphogenetic movements must take place before organogenesis occurs, even in regions such as the foregut and stomodaeum which develop in place after they form. It is also interesting to note that although the nervous system in these embryos showed typical differentiation into nerve-fibres and ganglion cells, no other evidence of differentiation of ectodermal tissues was found. Mesoderm and some gut cells were present, but their differentiation was slight.

The most interesting embryos, from the point of view of information about factors in differentiation and organization, were those in which a considerable degree of organization had occurred, but in which there were also many abnormalities.

The simplest kind of abnormality to explain was the distortion of spatial relationships within the embryo. The mechanical difficulties which prevent the shortening of the embryo result in the differentiation of the posterior spiracles in a position which they do not normally occupy at this stage. The movement of the whole blastoderm during treatment also leads to invagination of organ rudiments in abnormal positions. A shifting of blastoderm may also lead to abnormality in the position of the salivary glands which has been observed in some embryos (e.g. the embryo in Plate 2, fig. 11). Another factor of consequence is that the development of one organ in an abnormal position will force organs which form later into positions which they do not normally occupy.

Abnormalities in cellular differentiation are more difficult to explain satisfactorily. It is especially difficult to find the explanation of the high degree of involvement of the hypodermis, which was affected in every embryo in which organization had taken place. It is improbable that during treatment cells from which the superficial ectoderm will differentiate are preferentially destroyed; for the action of the ultrasonics is such that all cells in a particular region are involved. It is also highly unlikely that the superficial ectoderm develops entirely from cells at the posterior end. There seem to be at least two alternative explanations, neither of which can be proved because our knowledge of the processes of differentiation in mosaic eggs is so scanty. One possibility is that the development of the hypodermis depends on some special cytoplasmic factor or sub-microscopic cellular structure which is particularly easily destroyed by the treatment. The other, which merits more discussion, is that the great sensitivity of the hypodermis is the consequence of a disturbance of some inductive relationship.

Poulson (1945) has suggested, on the basis of abnormal differentiation in embryos deficient for the facet region of the X-chromosome, that there may be an inductive relation between the mesoderm and the ectoderm, the former acting as an inductor. His arguments are not very strong. If an inductive relationship of the kind proposed by Poulson does exist, it is not dependent upon differentiation of the mesodermal layer, for in one embryo (Plate 2, fig. 11) the mesodermal elements were completely disorganized although the hypodermis was better developed than in any other embryo studied.

More convincing evidence of inductive relationships between the two layers has been produced by the experimental work of Bock (1939, 1941) and Haget (1953a). Although this was performed on Neuroptera and Coleoptera, and not on Diptera, one may perhaps use it as a guide in interpretation. They find that the ectoderm (hypodermis) first exerts an inductive influence to which the mesoderm reacts. Later the mesoderm has an inductive influence on the development of the midgut (endoderm). Finally Haget in particular has emphasized the existence in beetles of an ‘intra-dermal’ induction process within the hypoderm, by which the development of the outlying parts is affected by their relation to the prothoracic region.

In seeking an explanation of the lack of differentiation of the hypodermis in the ultrasonic-treated embryos, it seems that one might appeal to this last type of relation, and suppose that the treatment had inhibited in some way an intradermal induction process which should normally occur within the ectoderm. It may be noted that the intradermal induction as described by Haget takes place between blastula formation and the end of gastrulation, so that one might expect a similar process in Drosophila to be strongly affected by ultrasonic treatment at the syncytial blastoderm stage.

In making this suggestion one is using processes found in one group of Insects, the Coleóptera, to throw light on phenomena occurring in another group, the Diptera. That the drawing of such parallels may be dangerous is suggested by another type of induction relation, that between the germ-cells and the mesodermal elements of the gonads. Haget (1953b) has shown that in Coleóptera the mesodermal part of the gonad develops even if the germ-cells are completely destroyed at the pole-cell stage. Aboim (1945) has claimed the same thing for Drosophila in which the pole-cells have been killed by ultra-violet light. However, in all the cases he illustrates there are germ-cells in the gonads, although they are dead and disintegrating. Since it is well known that dead tissues may exert evocating influences, his evidence cannot be taken as proving that the mesodermal gonad can differentiate in the absence of a stimulus from the germcells. In our material they certainly show no signs of such ability. The gonads are absent in many embryos in which the pole-cells were trapped in an abnormal region at the posterior end, so that it was impossible for them to be carried into the interior of the embryo. In some of these cases, where the extent of the damage at the posterior end was great, the cells which form the primordium of the posterior midgut were affected so that no invagination was formed by which pole-cells could be carried to the interior. However, in these embryos the polecells were so surrounded by the abnormal tissue that their movement seemed unlikely in any case.

On the other hand, we find positive evidence for the ability of the germ-cells to induce gonad formation. In two embryos (one of which is shown in Plate 2, fig. 12) the gonads formed in the anterior region just behind the mouth parts. In both instances, although the germ-cells had migrated into regions of mesoderm with which they are not normally associated, there was clear differentiation of the gonad sheath and the interstitial elements of the gonads which are formed from mesodermal cells. This would seem to indicate that the fate of some mesodermal cells is not completely determined. The possibility that a great shift in cellular blastoderm had occurred during treatment cannot be ruled out entirely in the case of one of these embryos, for other organs which are normally found anteriorly were in the posterior region. The orientation (anterior and posterior) of this embryo could be determined by the position of the chorionic filaments: there is also a characteristic thickened region at the posterior end of the chorion which may be used as an additional guide to orientation. The possibility of an error in interpretation as to anterior and posterior ends of embryos is therefore completely excluded. In the other embryo, however, the other organs developed in positions which were quite normal; in fact it was this embryo in which the most normal spatial relationships of organs in the anterior end, including the cephalo-pharyngeal apparatus and the chitinized structures of the anterior region, were found.

The abnormalities in tracheal formation and salivary glands may be explained from abnormalities in the separation of cells of the superficial ectoderm, since both organs develop from cells of the superficial ectoderm which begin to invaginate at about the seventh hour of embryonic life. The failure of head structures to develop can also be related to abnormalities in ectodermal differentiation, because the frontal sac, from which the chitinized structures of the head are derived, first forms as an invagination of superficial ectodermal cells in the anterior end of the embryo.

The slight abnormalities in organogenesis are also easily understood. Abnormal distribution of yolk is responsible for some of the abnormalities in segmentation which have been observed, while slight abnormalities in muscle differentiation or apodemal attachment account for others. The presence of a small clump of muscle-cells in the nervous system of one embryo may be the result of the inclusion of determined cells within an area destined to become something else. The cells subsequently developed independently into muscle-cells, although they were completely surrounded by nervous tissue.

The results obtained in treating embryonic stages with ultrasonics seem to justify the further use of ultrasonics in experimental embryology. There are indications that further work along these lines might reveal valuable information concerning the possibility of the existence of inductive relationships in Droso-phila embryos. It might also be possible to use ultrasonic treatment as a tool in solving the problem of the fate of the pole-cells in the embryo. It is known that some of the pole-cells form the gonads, but the fate of the others is unknown. It is also a matter for conjecture whether the pole-cells which enter the embryo before the formation of the ventral furrow form yolk-cells as suggested by Rabinowitz (1941), or form the gonads as Poulson (1950) has proposed. Poulson further suggests that the pole-cells which are moved to the interior in the posterior midgut invagination take part in the formation of the midgut only. Between the third and fourth hours of development the pole-cells lie in a shallow concavity formed by the cells of the posterior midgut anlagen. It might be possible, by treating embryos at this stage with ultrasonics, to move the pole-cells out of this concavity and prevent them from entering the embryo. If those polecells which have migrated previously are those which form the gonad, and the others are part of the midgut anlagen, as Poulson suggests, then the majority of embryos treated at this stage should have normal gonads, but the formation of the gut should be affected. Ultrasonic treatment of other insect embryos in which development is less determinate than Drosophila but in which determination is still a cytoplasmic function, such as in Camponotus which was studied and reviewed by Reith (1931), should give even more interesting developmental results.

The authors wish to thank Professor C. H. Waddington, F.R.S., for suggesting and providing facilities for the present investigation, for discussion, ‘and for advice in preparing this manuscript. One of us (S. J. C.) was the recipient of the Dorothy Bridgman Atkinson Fellowship, given by the American Association of University Women, and the work received the financial support of the Agricultural Research Council.

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    Abbreviations
     
  • A

    anterior

  •  
  • AMG

    anterior midgut

  •  
  • BLD

    blastoderm

  •  
  • BN

    blastoderm nucleus

  •  
  • BR

    brain

  •  
  • BT

    bristle

  •  
  • CEN

    centriole

  •  
  • CH

    Chorion

  •  
  • CHF

    chorionic filament

  •  
  • CHT

    chitin

  •  
  • CLN

    cleavage nucleus

  •  
  • CYT

    cytoplasm

  •  
  • DBN

    displaced blastoderm nucleus

  •  
  • G

    gut

  •  
  • GB

    germ-band

  •  
  • GO

    gonad

  •  
  • HEC

    head segment

  •  
  • HY

    hypodermis

  •  
  • MG

    midgut

  •  
  • MP

    Malpighian tubules

  •  
  • MS

    mesoderm

  •  
  • MUS

    muscle

  •  
  • NF

    nerve-fibres

  •  
  • NS

    nervous system

  •  
  • NUC

    nucleus

  •  
  • OES

    oesophagus

  •  
  • P

    posterior

  •  
  • PC

    pole-cell

  •  
  • PMUS

    pharyngeal muscles

  •  
  • SLG

    salivary gland

  •  
  • SP

    spiracle

  •  
  • SPA

    spiricular atrium

  •  
  • TR

    trachea

  •  
  • VAC

    vacuole

  •  
  • VM

    vitelline membrane

  •  
  • VNS

    ventral nervous system

  •  
  • YK

    yolk

Magnification: In figs. 2, 10, and 13-19 inclusive the scale represents 10μ. In all other figures it represents 100μ.

Plate 1

Fig. 1. Longitudinal section of an egg treated with ultrasonics at 0 3 watts/cm.2 1 hour after deposition, and fixed immediately after treatment, showing the curved outline of the disturbed regions. Cleavage nuclei are present. Note the absence of yolk granules at the periphery of the disturbed areas.

Fig. 2. Enlargement of the posterior region of an egg treated with ultrasonics at the syncytial blastoderm stage, and fixed immediately, showing the point of rupture of the syncytial blastoderm, and a typical disturbed area. Note the irregular dark mass, probably formed of fragmented yolk globules.

Fig. 3. Anterior region of embryo treated with ultrasonics at 0-3 watts/cm.2 during the syncytial blastoderm stage and fixed immediately. A disturbed region has formed at the anterior end, and yolk has flowed out of a small gap on the dorsal surface, distorting the shape of the embryo. Nuclei and cytoplasm in the disturbed region are abnormally distributed.

Fig. 4. Longitudinal section of embryo treated with ultrasonics at 0 ·3 watts/cm.2 during the syncytial blastoderm stage. Age at time of fixation, 6 hours. Only rudimentary gastrulation has taken place. The abnormal nuclei which form in the disturbed areas are clearly shown. Note the separation of yolk and cytoplasm in the abnormal region.

Fig. 5. Semi-longitudinal section of embryo treated with ultrasonics at 0·5 watts/cm.2 during the syncytial blastoderm stage. Age at fixation, 1912 hours. The anterior end is a cellular mass without any differentiation. The abnormal region occupies almost the entire posterior half.

Fig. 6. Longitudinal section of embryo treated with ultrasonics at 1· 2 watts/cm.2 during the syncytial blastoderm stage. Age at fixation, 19 hours. Differentiation of tissues, but no organization, has taken place.

Fig. 7. Longitudinal section of embryo treated with ultrasonics at 1·2 watts/cm.2 during the syncytial blastoderm stage. Age at fixation, 19 hours. Note the exaggeration in segmentation at the dorsal surface. No hypodermis is present ventrally. The atrium of the posterior spiracle is on the mid-dorsal surface, which indicates that shortening of the germ-band has not occurred. The central region is a mixture of cells of the midgut and muscle-cells.

Fig. 8. Median longitudinal section of the embryo shown in fig. 7. The mixture of muscle-cells, tracheal fragments, and gut cells in the central region is conspicuous. Involution of the head is abnormal, but there has been some organization of pharyngeal musculature.

Plate 1

Fig. 1. Longitudinal section of an egg treated with ultrasonics at 0 3 watts/cm.2 1 hour after deposition, and fixed immediately after treatment, showing the curved outline of the disturbed regions. Cleavage nuclei are present. Note the absence of yolk granules at the periphery of the disturbed areas.

Fig. 2. Enlargement of the posterior region of an egg treated with ultrasonics at the syncytial blastoderm stage, and fixed immediately, showing the point of rupture of the syncytial blastoderm, and a typical disturbed area. Note the irregular dark mass, probably formed of fragmented yolk globules.

Fig. 3. Anterior region of embryo treated with ultrasonics at 0-3 watts/cm.2 during the syncytial blastoderm stage and fixed immediately. A disturbed region has formed at the anterior end, and yolk has flowed out of a small gap on the dorsal surface, distorting the shape of the embryo. Nuclei and cytoplasm in the disturbed region are abnormally distributed.

Fig. 4. Longitudinal section of embryo treated with ultrasonics at 0 ·3 watts/cm.2 during the syncytial blastoderm stage. Age at time of fixation, 6 hours. Only rudimentary gastrulation has taken place. The abnormal nuclei which form in the disturbed areas are clearly shown. Note the separation of yolk and cytoplasm in the abnormal region.

Fig. 5. Semi-longitudinal section of embryo treated with ultrasonics at 0·5 watts/cm.2 during the syncytial blastoderm stage. Age at fixation, 1912 hours. The anterior end is a cellular mass without any differentiation. The abnormal region occupies almost the entire posterior half.

Fig. 6. Longitudinal section of embryo treated with ultrasonics at 1· 2 watts/cm.2 during the syncytial blastoderm stage. Age at fixation, 19 hours. Differentiation of tissues, but no organization, has taken place.

Fig. 7. Longitudinal section of embryo treated with ultrasonics at 1·2 watts/cm.2 during the syncytial blastoderm stage. Age at fixation, 19 hours. Note the exaggeration in segmentation at the dorsal surface. No hypodermis is present ventrally. The atrium of the posterior spiracle is on the mid-dorsal surface, which indicates that shortening of the germ-band has not occurred. The central region is a mixture of cells of the midgut and muscle-cells.

Fig. 8. Median longitudinal section of the embryo shown in fig. 7. The mixture of muscle-cells, tracheal fragments, and gut cells in the central region is conspicuous. Involution of the head is abnormal, but there has been some organization of pharyngeal musculature.

Plate 2

Fig. 9. Median longitudinal section of embryo treated with ultrasonics at 0·3 watts/cm.2 during the syncytial blastoderm stage. Age at fixation, 20 hours. Segmentation is present only in the dorsal posterior region. The anterior dorsal region and the ventral region are composed almost entirely of nervous tissue. The head segment has formed too far posteriorly, but the pharyngeal musculature is quite well organized. At the posterior end the embryo appears pushed in like a hollow ball to which local pressure has been applied. The posterior spiracles and spiricular atria may be seen in the interior.

Fig. 10. Enlargement of the posterior region of the embryo shown in fig. 9, showing the structure of the posterior spiracles. The structure of the hypodermis is also shown.

Fig. 11. Longitudinal section of embryo treated with ultrasonics at 1·2 watts/cm.2 during the syncytial blastoderm stage. Age at fixation, 18 hours. The mesoderm is disorganized. No differentiation of the gut from a primitive state has occurred, and the oesophagus and proventriculus (not shown) are very abnormal. Differentiation of the hypodermis is good, with the exception of the dorsal surface where it is lacking (failure of dorsal closure?). The salivary glands are displaced, and lie on the same side of the embryo, one more dorsally than the other. The presence of staining material in the lumen indicates that the functional activity of the glands has not been impaired. No mouth parts have formed, but the hypodermis in the anterior region is thickened. No true segments have formed.

Fig. 12. Semi-longitudinal section of embryo treated with ultrasonics at 12 watts/cm.2 during the syncytial blastoderm stage. Age at fixation, 19 hours. The hypodermis is completely lacking. The gonad has formed in the anterior region. Differentiation of the gut and of the visceral musculature is almost normal.

Fig. 13. Enormous resting-stage nuclei in an egg treated with ultrasonics at 12 watts/cm.2, 114 hours after deposition, and fixed 4 hours later. Note the centrioles.

Fig. 14. Spindle displaced to the side of a resting-stage nucleus, in an egg treated with ultrasonics at 1·2 watts/cm.2, 20 minutes after collection, and fixed 5 minutes later.

Fig. 15. A metaphase spindle split along its length in an egg treated with ultrasonics at 1·2 watts/cm.2,1 hour after deposition, and fixed 5 minutes later.

Fig. 16. Showing a spindle with centrioles and an absence of chromosomes, in an egg treated with ultrasonics at 1·2 watts/cm.2, 1 hour after deposition, and fixed 5 minutes later.

Fig. 17. A normal diploid metaphase figure in an egg treated with ultrasonics at 0 · 5 watts/cm.2, 114 hours after deposition, and fixed 5 minutes later.

Fig. 18. A polyploid metaphase figure (contrast with fig. 17), in an egg treated with ultrasonics at 1·2 watts/cm.2, 114 hours after deposition, and fixed 4 hours later.

Fig. 19. A polyploid metaphase figure, in an egg treated with ultrasonics at 114 hours after deposition, and fixed 4 hours later.

Plate 2

Fig. 9. Median longitudinal section of embryo treated with ultrasonics at 0·3 watts/cm.2 during the syncytial blastoderm stage. Age at fixation, 20 hours. Segmentation is present only in the dorsal posterior region. The anterior dorsal region and the ventral region are composed almost entirely of nervous tissue. The head segment has formed too far posteriorly, but the pharyngeal musculature is quite well organized. At the posterior end the embryo appears pushed in like a hollow ball to which local pressure has been applied. The posterior spiracles and spiricular atria may be seen in the interior.

Fig. 10. Enlargement of the posterior region of the embryo shown in fig. 9, showing the structure of the posterior spiracles. The structure of the hypodermis is also shown.

Fig. 11. Longitudinal section of embryo treated with ultrasonics at 1·2 watts/cm.2 during the syncytial blastoderm stage. Age at fixation, 18 hours. The mesoderm is disorganized. No differentiation of the gut from a primitive state has occurred, and the oesophagus and proventriculus (not shown) are very abnormal. Differentiation of the hypodermis is good, with the exception of the dorsal surface where it is lacking (failure of dorsal closure?). The salivary glands are displaced, and lie on the same side of the embryo, one more dorsally than the other. The presence of staining material in the lumen indicates that the functional activity of the glands has not been impaired. No mouth parts have formed, but the hypodermis in the anterior region is thickened. No true segments have formed.

Fig. 12. Semi-longitudinal section of embryo treated with ultrasonics at 12 watts/cm.2 during the syncytial blastoderm stage. Age at fixation, 19 hours. The hypodermis is completely lacking. The gonad has formed in the anterior region. Differentiation of the gut and of the visceral musculature is almost normal.

Fig. 13. Enormous resting-stage nuclei in an egg treated with ultrasonics at 12 watts/cm.2, 114 hours after deposition, and fixed 4 hours later. Note the centrioles.

Fig. 14. Spindle displaced to the side of a resting-stage nucleus, in an egg treated with ultrasonics at 1·2 watts/cm.2, 20 minutes after collection, and fixed 5 minutes later.

Fig. 15. A metaphase spindle split along its length in an egg treated with ultrasonics at 1·2 watts/cm.2,1 hour after deposition, and fixed 5 minutes later.

Fig. 16. Showing a spindle with centrioles and an absence of chromosomes, in an egg treated with ultrasonics at 1·2 watts/cm.2, 1 hour after deposition, and fixed 5 minutes later.

Fig. 17. A normal diploid metaphase figure in an egg treated with ultrasonics at 0 · 5 watts/cm.2, 114 hours after deposition, and fixed 5 minutes later.

Fig. 18. A polyploid metaphase figure (contrast with fig. 17), in an egg treated with ultrasonics at 1·2 watts/cm.2, 114 hours after deposition, and fixed 4 hours later.

Fig. 19. A polyploid metaphase figure, in an egg treated with ultrasonics at 114 hours after deposition, and fixed 4 hours later.