Waddington1956 8

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Waddington CH. Principles of Embryology (1956) The MacMillan Co., New York

   Principles of Embryology (1956): Part 1 - 1 The Science of Embryology | 2 The Gametes | 3 Fertilisation | 4 Cleavage | 5 The Echinoderms | 6 Spirally Cleaving Eggs | 7 The Ascidians and Amphioxus | 8 The Insects | 9 The Vertebrates: The Amphibia and Birds | 10 The Epigenetics of the Embryonic Axis | 11 Embryo Formation in Other Groups of Vertebrates | 12 Organ Development in Vertebrates | 13 Growth | 14 Regeneration | 15 The Role of Genes in the Epigenetic System | 16 The Activation of Genes by the Cytoplasm | 17 The Synthesis of New Substances | 18 Plasmagenes | 19 The Differentiating System | 20 Individuation - The Formation of Pattern and Shape | References
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Chapter VIII The Insects

The great group of insects contains an enormous range of different forms, and any treatment of insect development which can fit into the framework of this book must be an extremely summary and simplified one. There is not only a very wide range of different types of embryonic development to be covered, but our knowledge of the phenomena of insect metamorphosis is considerable, and, although we have neglected this problem in the annelids and echinoderms, it seems desirable to give some account of it in insects. As is well known, there are all gradations in the intensity of the changes involved in metamorphosis. In some primitive insects (the Ametabola), the larval form develops gradually into the adult, with no sudden or marked change. There are other more complex types, in which the wing buds may be exposed in the larval stages (Exopterygota) or concealed beneath the surface until the time of metamorphosis (Endopterygota); and again the larva (in this case often known as a ‘nymph’) may be directly transformed into the adult (Hemimetabola) or there may be a pupal stage intercalated (Holometabola). In the extreme type, the Holometabola, the adult organism may have almost no immediately obvious similarity to the larval; the life-history comprises two very dis~ tinct developmental systems. Since there will be no space to treat all the intermediate conditions, we shall find it convenient to deal first with the embryonic development, by which the larva is produced, and then to pass on to the processes of metamorphosis, particularly in those types in which they are most intense and far-reaching.

1. Embryonic development

There are many types of insect eggs (Review: Johannsen and Butt 1941), but they are all variations on a fundamental plan which is at first sight rather unlike that of any of the other eggs described in this book (although related to those of other arthropods, which we shall not consider). Their most obvious characteristic is that the yolk, instead of being concentrated at the vegetative end of the cgg, is more or less uniformly distributed throughout the whole central region of it; whence the eggs are often referred to as ‘centrolecithal’ in contrast to the ‘telolecithal’ eggs of other groups. Around the central mass of yolk, there is always a cortex, or peripheral sheet of cytoplasm, which may be quite thin, but is sometimes fairly thick. In recent years, the importance of the cortex in other eggs, such as those of the echinoderms, the spiral-cleaving forms, and the Amphibia, has come to be recognised, and with this recognition, the conditions in the insect egg begin to seem less peculiar than they had done previously. Instead of their organisation being fundamentally different from that of other groups, we sce that they differ only quantitatively, in that their cortical plasma is rather thicker than usual, and their yolk more evenly distributed within the interior cytoplasm.

Insect eggs often exhibit an obvious bilateral symmetry, the dorsoventral plane being built into them during their formation in the ovary. They also tend to be provided with rather rigid external membranes, through which a micropyle leads the sperm in to the egg. The vitelline membrane, formed by the egg itself, is often partly chitinised and highly impermeable to most solvents, a circumstance which makes fixation difficult and renders the eggs troublesome objects for detailed study.

Although as we have seen the structure of the insect egg is not entirely dissimilar to that of other groups, it is nevertheless peculiar enough to cause profound modifications in the cleavage. Insect eggs, in fact, do not cleave at all in the normal sense of the word. After fertilisation the gamete-nuclei conjugate somewhere in the neighbourhood of the micropyle, and the zygote nucleus which is thus constituted divides a considerable number of times without any accompanying division of the main mass of egg cytoplasm. Each nucleus, however, is surrounded by a small region of relatively non-yolky cytoplasm and at each nuclear division, this divides into two. The nuclei, accompanied by their patches of cytoplasm, move away from one another as though repelling each other, and usually go through a stage in which they are arranged roughly as a hollow sphere, or at least a hollow shape more or less conformable to the outline of the whole egg (Fig. 8.1).

After some time, most of the nuclei reach the cortex and pass into it, their cytoplasmic halos fusing with it. It is not until the cortex becomes populated with nuclei that cell boundaries make their appearance, and they form only in the cortex itself, which thus becomes converted into an epithelium enclosing the main mass of yolk (which still contains a few nuclei, whose later function is to take part in the digestion of the nutritive materials). The enclosing epithelium is known as the blastoderm, and it is from this that the embryo develops.

The first sign of the embryo is a thickening of the blastoderm. This may at first be double, so that there are two thicker strands, with many nuclei, rather like the embryonic bands which form in an annelid which develops without any larval stage, such as Tubifex (p. 96). Soon, however, the bands come together and form a single elongated thickening, the socalled ‘germ-band’. The midline of the germ-band is the mid-ventral line of the final animal; in insects, it is the ventral side, not the dorsal, which plays the dominant role.

Figure 8.1

Generalised diagram of insect embryonic development: (a) is a section showing the cleavage nuclei, which collect at the surface to produce a blastoderm (b). The number of cells increases, and two densely ageregated groups appear (c). These come together and fuse to form a single germ-band (d).

Along this mid-ventral line, the germ-band folds downwards into a groove whose edges come together to constitute the ectoderm, while the floor of the groove disappears below the surface, as a lower layer or ‘hypoblast’, which eventually gives rise both to mesoderm and to the endoderm of the mid-gut. At the anterior and posterior ends of the germband, pockets are pushed in along the length of the embryonic body, forming respectively the mouth aid anus, or, more correctly, the stomadeum and proctodeum. From these, more endoderm is produced, to develop into the foregut and hindgut.

There is one other important group of internal cells which may be mentioned. In many insects, (particularly in the ‘determinate’ type to be mentioned below), the cells at the most posterior end of the blastoderm have a special character, distinguishing them histologically from the rest. They are known as the ‘pole cells’, and they later migrate into the gonads and become the source of all the later-formed gametes. If they are removed, by cauterising or otherwise killing them at an early stage, completely sterile individuals may be produced, and it seems that in some forms at least they are the only cells of the embryo which are capable of differentiating into gametes. The whole developmental sequence of mother- and daughter-cells, from the pole cells to the final gametes, is sometimes known as the ‘germ-line’, a term to be carefully distinguished from the ‘germ-band’ which, as we have seen, is used for the blastodermic thickening from which the embryo arises.

While the lower layers of the embryo are forming in the way described above, the germ-band is becoming transversely segmented, and from each of the anterior segments, outgrowths protrude and grow into the cephalic and thoracic appendages of the larva. All the main organs of the embryo are then at least indicated, though they still have much histological differentiation to carry out before they are fully developed. By the time the main blocking-out of the embryo is complete, the germ-band may still represent only the ventral surface, the dorsal side being occupied by the mass of yolk. At some stage, often quite a late one, the body wall is completed by the outward growth of the two lateral edges of the germ-band, which eventually meet and fuse at the mid-dorsal line. This finishes the laying down of the embryo proper.

(e),( J), (g) show the outline of the germ-band, with the longitudinal furrow in which the mesoderm is invaginated (M.F.) and the transverse furrows delimiting the segments: the arrow at the side marks the first intersegmental furrow, which is between the most posterior head-segment (second maxillary) and the most anterior thoracic; (h) and (7) show sections through the stages of (e) and (g) at the level of the arrow to show the invaginating mesoderm (dotted) becoming covered by ectoderm (lined). The next stage is shown in surface view (j) and longitudinal section (k); the stomodeum (foregut) and proctodeum (hindgut) are pushed inwards from the surface, and from the former endoderm arises and will develop into the midgut; the appendages are beginning to appear on the segments of the embryo. Figures (/) and (m) are transverse sections of rather ‘later stages to show how the edges of the embryonic area grow round to enclose the yolk and thus provide the dorsal surface of the completed embryo, which is drawn in (1) (After Seidel 1936.)

In some insects, more or less elaborate extra-embryonic structures are also developed. There are often membranes which cover the embryo and presumably help to protect it from outside influences. These are derived from parts of the blastoderm peripheral to the germ-band, by the formation of folds which eventually meet and fuse above the embryo, leaving an outer layer (or serosa) and an inner (or amnion). There is another and most peculiar phenomenon to be mentioned, which however plays no part in the actual formation of the embryonic organs, namely the socalled ‘blastokinesis’. This is the name for a series of movements by which the germ-band may at some stage be dragged from the surface right into the middle of the yolk mass, only to emerge on to the surface again later. The most strongly developed blastokinetic movements occur in species in which the embryo is small compared with the size of the whole egg, but even in forms in which the embryo occupies almost the entire available space, there are often considerable shiftings of the blastoderm as a whole (cf. Fig. 8.2).

As was pointed out above, there is within the insect kingdom a very large range of variations around the generalised type which has just been described. These variations fall into a series, from an ‘indeterminate type’ at one end to a ‘determinate type’ at the other (Seidel 1936). The indeterminate type includes the eggs of some of the more primitive groups of insects, such as Orthoptera and Odonata; it is characterised by eggs which are usually rather large and often elongated, provided with only a thin cortex, and developing an embryo which is small in relation to the egg. The name ‘indeterminate’ is used because very considerable regulation is possible during the early stages of development. In the determinate type, regulation is very slight or altogether absent. The most typical representatives of this group are the Lepidoptera and Diptera, whose eggs have a thicker cortex, which is often regionally specialised even before fertilisation. The embryo normally occupies the whole, or at least the greater part THE INSECTS 123

of the space available to it. Between these two extremes there are various intermediate types of eggs, of which those of the Hymenoptera and Coleoptera are the best known.

An experimental study of development in the related group of Arachnids has recently been published (Holm 1952).

2. Experimental analysis of some types of insect development

It is the indeterminate type, with its capacities for regulation, which allows us most easily to gain an insight into the epigenetic system of the insects. We shall therefore begin by considering a typical representative of this type, the dragon-fly (Odonata) Platycnemis pennipes (Seidel 1929, 1936).

The cleavage of the nuclei, their repulsion from one another, and the eventual formation of a blastoderm covering the whole surface proceed in an absolutely typical manner (Fig. 8.2). A germ-band then appears, in the form of a region of the blastoderm which is thicker and contains a higher concentration of nuclei. It at first shows some signs of doubleness, but soon shortens somewhat and becomes a single area except in its most anterior part, where there are two lobes which will eventually develop into the head. The next stages of differentiation—that is, the further thickening of the germ-band, the folding inwards of the lower layer, the formation of transverse segments and the appearance of the appendages—all begin in a region which lies a short distance posterior to the head, in an area which later becomes the anterior thorax, and they spread both anteriorly and posteriorly from there until they affect the whole germ. This region, where development is visibly most advanced, is known as the Differentiation Centre; we shall see that it has important physiological functions as well as being morphologically in the lead. During the development of the embryo, a considerable blastokinesis occurs, the whole germ being at first folded deep into the yolk, and then emerging again, at the same time twisting around its longitudinal axis, so that it eventually lies with its dorsal side against the same part of the egg membrane as was originally in contact with its ventral face. The dorsal wall is not actually completed until after these blastokinetic movements have ceased.

The early stages of the embryo have very considerable powers of regulation, and two complete embryos can sometimes be produced by a single egg if, in some way or another, the developing system can be broken into two effectively separate parts. A mechanical bending of the egg is in some cases sufficient to split the yolk into two parts, and if this happens at an early stage of the dispersion of the nuclei, each part forms its own complete embryo. Owing to the extensive movements which take place during development, the relations of these twins to one another may become very peculiar. In one case described by Seidel, a slightly oblique fold had separated the egg into a larger and a smaller portion. Each developed an embryo, and the two twins must at first have lain back to back. As each spread out from the original ventral rudiment to close in and cover their dorsal sides, the larger engulfed the smaller and, moreover forced it to roll up the wrong side out. Thus the egg finished by containing a large embryo which enclosed a smaller twin which was inside-out, surely one of the most remarkable arrangements ever produced (Fig. 8.3).

1 General reviews: Seidel 1952a, 1953; Richards and Miller 1937, Krause 1939.

FicureE 8.2

Development of the dragonfly Platycnemis. Figures a, b, c, and d are sections showing the first three divisions of the nucleus, and the spreading of the daughter nuclei through the egg cytoplasm (the two sister nuclei from a division are joined by a dotted line). In e the nuclei are beginning to reach the surface; f, g and h are surface-views showing the multiplication of the nuclei to form a blastoderm and their aggregation to produce the germband (G.B.). In i the germ-band is being pulled into the interior of the egg; at the stage of Figure j nearly the whole embryo (dotted) lies internally. It is then pulled out again, at the same time rotating around its longitudinal axis (k). Note that in its final position (J) its head (H) is again towards the pointed end of the egg. (After Seidel 1929.)

More insight into the causal processes of development is gained from experiments in which the egg is divided in two transversely. This can be done either by killing off one end by burning with a microcautery, or better by constricting the egg with a loop of hair so that it is divided into two parts. By using these methods, Seidel showed that during the dispersal of the cleavage nuclei there is a small region at the posterior end which is essential for all further development, since if more than a very small fraction of this end is completely removed, the egg forms a blastoderm but never proceeds to develop a germ-band or embryo. This essential posterior region was named the ‘formation centre’ (Bildungszentrum’ in German). Its activity is almost certainly concerned with the production of a diffusible chemical, since if the egg is constricted by a hair which is not pulled completely tight but leaves a small channel through which diffusion could take place, the formation centre can still be effective and the anterior part develop. This only occurs, however, if the constriction is made after the formation centre has become populated with the dispersing cleavage nuclei. If, at an earlier stage, one makes a partial constriction which is loose enough to permit diffusion of chemical substances but too tight to allow nuclei to pass, the formation centre can never become nucleated, and it appears to be quite ineffective, since no embryo is developed. One must conclude that the formation centre consists of some specialised cytoplasm at the posterior end, but that it requires to be activated by the passage into it of a cleavage nucleus (together with the accompanying cytoplasmic halo); after being activated, the formation centre gives out a chemical substance which diffuses forwards and enables the main part of the egg to develop (Fig. 8.4).

Ficure 8.3

An internal twin in Platycnemis, following a longitudinal split in the egg . during the cleavage stages. Both longitudinal halves produced an embryo, and as the larger germ-band grew round dorsally to enclose the yolk (direction of thick arrows) it reversed the dorsal closure of the smaller one (which should have grown round in the direction of the thin arrows). In (a) the internal embryo is shown dotted. In the transverse section (b) note that the internal embryo is inside out, with the appendages (Ap. 1) inside and the nerve-cord (Nc. 1) outside the hypodermis (Hy. 1); the organs of the external embryo are at Ap. 2, Nc. 2 and Hy. 2. (After Seidel 1936.)


The operation of the Formation Centre. Ifa very small part of the posterior

of the egg is constricted off at an early stage (a), an embryo can develop (b);

but if the constriction lies a little further forward (c) no embryo forms (d).

After the formation of the blastoderm, however, an embryo is formed even

if the constriction lies well forward (e), since the Formation Centre has by that time completed its action. (After Seidel 1929.)

Seidel was able to alter the regular process of dispersal of the cleavage nuclei, either by killing one with localised ultra-violet irradiation or by partially ligaturing the egg for some time so that the dispersal had to occur in an abnormal space. He could in this way cause the formation centre to be invaded by a nucleus other than that which would normally have reached there; and he showed that any nucleus—or at least any of the 128 which are formed in the first seven divisions—is pecdutte to activate the centre.

The chemical which diffuses forwards from the formation centre does not act directly on each separate part of the egg. It sets going the differentiation centre, which is not only morphologically distinguishable, as the site of the first steps in the development of the germ-band, but is also the second centre of physiological activity. Once it is activated by the substance from the formation centre, it becomes a focus around which the embryo is organised. The region over which its activity extends can also be broken by constricting the egg with a hair loop, but in this case the continuity is much more easily disrupted, and even a fairly loose constriction is enough to inhibit the passage of the differentiation centre’s influence. Thus if a loose loop is tied round the egg posterior to the differentiation centre, between it and the formation centre, an embryo develops only in the anterior region; if the loop lies anterior to the centre, the embryo lies wholly posterior to the loop, while if the constriction is located actually within the differentiation centre, twin embryos will form, one in each part (Fig. 8.5).

Ficure 8.5

The action of the Differentiation Centre in Platycnemis. If a loose constric tion is made in front of the Centre (a) the embryo forms wholly posterior

to it. If the constriction is behind the Centre, the embryo lies wholly in front

of it (c). If the constriction is at the actual site of the Centre, this may be

divided and embryo formation take place both in front and behind (b). (After Seidel 1938.)

The ease with which the influence of the differentiation centre is interrupted shows that it is not transmitted by a diffusing chemical. Seidel. came to the conclusion, in fact, that the activity of the centre is fundamentally mechanical, and consists in a contraction of the yolk, which leaves a space into which the cells of the blastoderm migrate, thus forming the thickened region which becomes the germ-band. This contraction 128 PRINCIPLES OF EMBRYOLOGY

normally begins in the middle of the differentiation centre, and spreads out in each direction from there; a comparatively slight mechanical abnormality, such as is caused by a loose constriction, will either inhibit the spread of the wave of contraction, or split into two. Thus the central yolk mass of the egg, which might at first sight be taken to be a merely passive supply of nutrients, is actually not so at all, but by its contraction plays an essential role in the epigenetic system which gives rise to the embryo.

After the action of the differentiation centre is completed, the various regions of the embryo seem to be fairly definitely determined, and only very slight regulation remains possible.

In the period since Seidel carried out his pioneer work, a number of other insect eggs have been found to follow a more or less ‘indeterminate’ type of development; that is to say, they are capable of some degree of regulation, and provide evidence of a sequence of epigenetic processes.

In the Coleoptera (beetles) it was shown long ago that there are special ‘pole cells’ at the posterior end of the blastoderm, and that if these are killed, no reproductive cells are produced by the embryo. In other respects, however, some regulation is possible. In the mealworm Tenebrio (Ewest 1937), there is a first stage which lasts from fertilisation till the sixth cleavage (sixty-four nuclei). If, during this stage, a posterior region (about 20 per cent of the total length) is removed or cauterised, the movement of the nuclei into the cortex ceases and not even a beginning of development occurs; but if a similar defect is made after the 64-nuclei stage, blastoderm formation proceeds although no embryo will develop. Thus this posterior region must exert some action in the very early cleavage stage, and it therefore seems to be similar to a formation centre, although it does not seem to require activating by the presence of a nucleus, as does that of Platycnemis.

In another beetle, Leptinotarsa (the “Colorado beetle’), Haget (1953), in a very thorough and technically accomplished study, could discover no evidence for the existence of a formation centre active in the early stages. He is inclined to doubt whether Seidel’s evidence in Platycnemis really proves the existence of such a centre, but in this he seems perhaps unduly sceptical.

Most recent workers have entirely confirmed the existence of a differentiation centre, lying further anteriorly, and operating some time after the beginning of development. Moreover our knowledge of the successive steps in the action of this centre has been greatly extended recently, particularly by the work of Bock (1942) on the neuropteran Chrysopa, Haget (1953) on Leptinotarsa, and Krause (1953) on the grasshopper Tachycines. All these forms give evidence of a series of inductive interTHE INSECTS 129

actions between the various germ layers, and thus suggest similarities between insect development and the phenomena which we shall find in the development of the vertebrates. According to the available information, there are certain differences in the epigenctic reactions in the various forms, but it seems not improbable that these may tend to disappear as the territory opened up by the recent pioneering work becomes more fully explored.

In all three forms, Chrysopa, Leptinotarsa and Tachycines, the differentiation centre begins to be active in the anterior part of the germ-band, which will later develop into the prothoracic region. Its presence is denionstrated by the fact that parts of the embryo removed from contact with the centre do not continue differentiating. Haget shows that in Leptinotarsa, the centre is at first localised in a small region, and gradually spreads in all directions (Fig. 8.6). In Tachycines, Krause finds that the first

FiGure 8.6

The increase in size of the ‘Differentiation Centre’ in Leptinotarsa, from 18

to 27 hours of age. If the embryo is cut in half through the black region, both

parts develop partial embryos; if the cut is in the dotted region, the larger

part forms a partial embryo and the remainder a germ-band which fails to

develop further; while if the cut is in the white areas, only the larger part shows any signs of development. (From Haget 1953.)

result of the action of the centre is a tendency for the mesoderm to be invaginated. If, following injury, the mesoderm does not form, the ectoderm fails to develop; and Krause concludes that it is the mesoderm which endows the ectoderm with the capacity to differentiate. He points out, however, that the manner in which the ectoderm develops (i.e. the organs it forms) is not dependent on the nature of the mesoderm underlying it. In fact, once the ectoderm has been set going, the main inductive influence goes the other way, the character of the ectoderm determining what type of mesoderm shall be produced. Bock and Haget quite independently also obtained clear evidence for this induction of mesodermal tissues by the ectoderm, in Chrysopa and Leptinotarsa. They both found, however, in contradistinction to Krause, that the ectoderm could self-differentiate histologically from an early stage quite independently of the presence of mesoderm, although in Chrysopa its morphogenesis is abnormal if it is not underlaid by mesoderm. Haget in particular has studied the gradual acquisition by the various regions of the ectoderm of the capacity for selfdifferentiation, and the accompanying loss of its ability to regulate. He has shown that the process is dependent on an influence which spreads from the differentiation centre through the sheet of ectoderm; he speaks of it as a process of ‘intra-dermal induction’, and it may be compared with the ‘individuation’ or ‘regionalisation’ by which various organs become localised within the sheet of invaginated mesoderm in the vertebrates (Fig. 8.7).


Results of cauterisation of the endo-mesoderm or ectoderm in Leptinotarsa. Figure 1 shows the whole median plate (pl.m.) of the germ-band destroyed. In the resulting embryo (Figure 2) the ectodermal organs are present (ir. tracheae, g. ganglion, app. appendages) but there are no mesodermal or endodermal tissues. In Figure 3 one of the lateral plates of the germ-band (presumptive ectoderm) has been cauterised. The embryo formed (Figure 4) lacks ectodermal organs in the cauterised region, and the endodermal and mesodermal organs are also absent where the ectoderm is defective. Figure 5 shows a section through a normal embryo (p. end., endodermal lining of gut, m., muscle, im., midgut, tmi., mesodermal tunic of midgut). (After Haget 1953.) THE INSECTS 31

It is a most remarkable fact that in the insects it is the ectoderm which takes the lead in the determination of development, while in vertebrates this function belongs to the inner layers, endoderm and particularly mesoderm. Whether this has any connection with the ‘upside-down’ morphology of the adult insect as compared with the vertebrate (ventral nerve cord instead of dorsal) is an intriguing question. One might, perhaps, think that it was more likely that conditions in the insects should be similar to that in annelids; but again, in Tubifex as we have seen, the development of the ectoderm is dependent on influences from the mesoderm (p. 99). There seems to be no close parallel in other groups to the situation found in the insects.

Haget has also evidence of a later inductive process, by which the mesoderm, after submitting to the influence of the ectoderm, itself induces the differentiation of the gut endoderm.

The Hymenoptera (Fig. 8.8) are a group which can be considered as intermediate between the indeterminate and fully determinate types, although somewhat nearer the latter. The main studies in this group have been on an ant, Camponotus (Reith 1931), and the bee, Apis (Schnetter 19344, b). In the former, the cortex is at fertilisation more or less uniform in thickness over the whole surface of the egg, although perhaps slightly thicker on the ventral side. As early as the stage of nuclear cleavage, however, it becomes differentiated into five zones, by the flow of internal cytoplasm to particular parts of the surface. This earliest stage of differentiation is suppressed if the posterior end of the egg is destroyed by cauterisation, which perhaps indicates that it is controlled by something analogous to a formation centre. The most important of the zones is that from which the germ-band arises, which may be compared to the differentiation centre. Cauterisations in the second half of the cleavage period allow the differentiation of the zones to proceed, but the germband zone may be shifted somewhat from its normal position. After the cortical zones have once been fully developed, no further regulation seems to be possible; both the formation centre and the differentiation centre have finished their work before the blastoderm is developed. (It is worth remarking that two of the cortical zones in the ant are concerned with the uptake of symbiotic bacteria, which seem to be always present in the egg, and which finally arrive in the midgut; their function is obscure.)

Ficure 8.8

a, b, Longitudinal sections through eggs of the ant Camponotus: a, shortly after laying, with cortex more or less evenly spread over the whole surface except for a slight thickening at the posterior end; b, 6-12 hours later, showing the differentiation of regions within the cortex. P is the collection of granules which will pass into the pole-cells (future germ-cells); C.N., cleavage nuclei. (After Reith 1931.) c, d, Longitudinal sections of eggs of the bee, Apis: c, just after laying; d, stage with 512 nuclei. C, cortex, C.Z.; central zone; D.C., Differentiation Centre; M.p., maturation plasma ; * sperm nucleus. (After Schnetter 1934).

In another hymenopteran, the bee Apis, development is even more precocious, since there are clearly marked cytoplasmic regions in the egg before fertilisation. The cortex is thicker on the ventral side, and in the anterior of this side there is a special collection of cytoplasm in which the maturation of the egg nucleus occurs. Slightly posterior to this is the region of the greatest diameter of the egg, and in this neighbourhood the cortex is thicker and there is more internal cytoplasm mingled with the yolk. A column richer in cytoplasm and poorer in yolk extends down the whole centre of the egg. This structure of the egg clearly affects the migration of the cleavage nuclei, which move into an elongated oval. The shape of this is not quite symmetrical, since the nuclei reach the surface first in the anterior ventral region, which can be considered as the analogue of the differentiation centre. As in Platycnemis, this seems to be the focus of a contraction of the internal material of the egg, but in this case the contraction is concerned not so much with the formation of a simple thickening of the blastoderm, but rather with the infolding of the inner layer, which occurs at a slightly later stage. There is in the bee as yet no evidence for the operation of a formation centre preceding the differentiation centre, but if small portions of the anterior part of the egg are removed by cauterisation, the differentiation centre shifts slightly posteriorly and complete but dwarf embryos are formed. By the early THE INSECTS 133

blastoderm stage, the position of the centre is fixed, but a small amount of regulation is still possible within individual organs; that is to say, if an egg in this stage is ligatured, each end forms only part of an embryo, but each of these parts consists of a certain assemblage of complete regions, ie. a complete anterior head, or complete jaw region, complete thorax or complete abdomen. At a slightly later stage, even this degree of regulatory power is lost, and ligatured eggs develop ina completely mosaic way, producing part thoraces or part abdomens exactly according to the location of the ligature.

In fully determinate eggs, such as those of Diptera, almost completely mosaic development occurs from the very earliest stages, and after cauterisations or ligaturing, no sign of regulation is found. Dwarf embryos have, however, been produced by strong centrifuging which shifts the cortex (Pauli 1927) and it appears that it is in the cortex that the forerunners of the embryonic organs are located. Small drops of the internal cytoplasm can in fact be removed (by pricking and allowing them to exude) without causing abnormalities in development (Howland and Sonnenblick 1936) (Fig. 8.9).


a, Normal embryo of the dipteran fly Calliphora; b, embryo formed after

constriction of the egg at the 16-cell stage, showing no sign of regulation;

c, shortened and partially regulated embryo following strong centrifugation of newly fertilised egg. (After Pauli 1927.)

A little information on the epigenetics of the dipteran Drosophila, which is so important for genetical studies, is beginning to be produced by less conventional methods. Thus Yao (1950) has investigated histochemically the distribution of acid and alkaline phosphatase, and finds that the latter makes its appearance in the future thoracic region of the germ-band some time after the mesoderm has invaginated, and gradually spreads throughout the ectoderm, by a process which he compares with the infective transmission of pigment formation in guinea-pig skin (p. 397). This reminds one strongly of the Differentiation Centre and its behaviour as described by Haget in Leptinotarsa.

Another method which has recently been employed is the application of ultra-sonics for the purpose of stirring round the internal contents of the egg (Selman and Counce, 1953, 1955). In cleavage stages this treatment may cause many types of cytoplasmic disturbance. If it is applied when the nuclei have arrived at the surface, preparatory to forming a cellular blastoderm, it may prevent the gastrulation movements, so that the tissues differentiate fairly normally but in the positions which they have before gastrulation occurs; or it may shift around the various cellular regions, in which case the organs are later found in quite unusual positions. In the latter case, there is usually some abnormality or deficiency in tissue differentiation; in particular the hypodermis tends to be badly developed. There are suggestions that some of these abnormalities of differentiation are consequences of disturbances of inductive relationships, but in most cases the evidence is not clear. Selman and Counce find, however, that gonads may develop in quite unusual parts of the body to which the germ cells have been transported, and argue that these cells induce the mesoderm near them to develop into the gonad sheath. (In Leptinotarsa Haget [1952] finds that the gonad mesoderm can differentiate in the absence of the germ-cells, and the same is probably true of Drosophila [Aboim 1945] though here the evidence is not entirely convincing; however even if it is accepted, this would not disprove the suggestion that the germ-cells can also exert an inductive influence.)

The most important aspect of the embryology of Diptera, however, is not its analysis by the operative methods of normal experimental embryology, but the opportunity provided by the enormous wealth of genetical material available in Drosophila to study the action of genes in early development. The pioneer in this work was Poulson (1945, 1950), and the most recent work that of Ede (1954) and Counce (1955). The principle which has been followed is to study the development in cases where the genetic situation causes death before hatching (in which case one can be certain that something fairly drastic is going on). A considerable number of factors have now been investigated, and the most important general points emerging seem to be the following.

Genes may be active at a very early stage of development. Poulson (1945) found that the absence of the whole or the greater part of the X chromosome causes disturbances of the cleavages and the migration of the cleavage nuclei. Ede found that certain sex-linked genes, which may be point-mutations, or may be very small deficiencies, may have the same THE INSECTS 135

effect; thus it is not only large amounts of chromosome, but on the contrary individual genes which are active at this stage.

A considerable number of genes are found to affect the process of gastrulation, which involves extensive movements of the blastoderm. This is clearly a delicately balanced process, which can easily go wrong; it is an ‘epigenetic crisis’, that is, a time at which minor abnormalities which have occurred earlier suddenly produce far-reaching and drastic effects.

Certain genes cause considerable abnormalities in tissue differentiation. These seem to be of two kinds; general retardation or impairment of differentiation, the cells remaining rather embryonic in character; or the switching of cells which should develop into one type of tissue into some other of the characteristic types. An example of the latter. effect is the observation that in several cases an abnormally large proportion of the hypodermis develops into neural tissue, leaving little or none to form skin. This might be the result of a disturbance of an intra-dermal inductive process within the hypoderm, of the kind postulated by Haget (p. 130).

These facts make clear the importance of chromosomal genes in the early developmental processes. Counce (1954) has recently studied in detail some stocks in which the importance of the cytoplasm becomes obvious. These are “female-steriles’, that is to say, races in which females homozygous for a given gene produce eggs which do not develop properly (see also Beatty 1949). This failure must be due to abnormalities in the cytoplasm formed under the influence of these genes in the ovarian tissues of the mother. In one of the genes studied, deep orange, the abnormality is similar to the first kind mentioned above, in that it is manifested in the early cleavage divisions. Another, fused, affects some of the elongation movements concerned in gastrulation (Fig. 8.10), while a third brings about an arrest of differentiation during a certain period of later embryogenesis. There is thus a considerable variety of processes which such cytoplasmic factors may influence.

The nature of the cytoplasmic factors is not at all clear. They may perhaps be ‘plasmagenes’ (p. 387), that is to say, have some power of reproduction; but it is unnecessary to make this assumption, since no overall growth has occurred by the time they begin acting. A very interesting fact, however, is that their adverse effects can be to a large extent overcome by the normal allele of the locus. If eggs from a female homozygous for one of these female-steriles (e.g. fused) is fertilised by sperm carrying a normal X chromosome, complete development may occur, and even if it does not do so, the egg develops more normally than otherwise. Some alleviating effect of this kind, though less in degree, is found even when such eggs are fertilised by chromosomes bearing a Y chromosome (which, if they could develop fully, would produce males). In this case the alleviation is probably caused by supernumerary X-bearing sperm, which enter the egg cytoplasm, but later degenerate, since they do not unite with the egg nucleus; it is known that in Drosophila it is common for five or six sperm to penetrate the egg.


Figure 8.10

Diagrammatic longitudinal section of Drosophila embryo developed from an egg of a fused mother mated to a fused male: displacement of organs due to faulty gastrulation movements. (From Counce 1955.)

We are thus beginning to get, in Drosophila, some insight into the interaction of genes and cytoplasm in early development, a subject which is obviously of the greatest importance, but in which much remains to be done. The study of hybrid merogons in Amphibia (p. 358) and echinoderms belongs to the same general sphere of interest, but in those organisms we cannot, as yet, investigate the effects of individual genes.

3. The transformation of the embryo into the adult

All insects, as they grow, pass through a series of moults, in which the external cuticle is shed and a new cuticle formed around the enlarging insect. As the moults proceed there are changes in the organisation of the THE INSECTS 137

animal, which finally becomes an adult. In some types, these changes are fairly gradual (‘hemimetabolous’ insects with an incomplete metamorphosis), in others there are first a series of larval stages in which little alteration occurs except increase in size, but these are followed by a rather sudden radical reorganisation by which the adult is produced (‘holometabolous’ insects with a complete metamorphosis). The period during which the metamorphosis occurs is known as pupation, and the pupal form usually differs considerably both from the larval and the imaginal phases of the life-history.


Diagram of the hormonal control of moulting and metamorphosis in insects. The letters above refer to the relevant larval organs: br. the brain; nsc, the neurosecretory cells; R.G., the ring-gland (in Diptera) consisting of cc, the corpus cardiacum, ca, the corpus allatum, and Ic, the lateral cells, which probably function as the prothoracic gland, which in other forms lies some distance from the corpus allatum, and is indicated as ptg; ib is an imaginal bud. The letters below refér to the active principles: N, the nervous connection to the corpus allatum; A, the activator passing from the neurosecretory cells to the prothoracic gland; GMH, the growth and metamorphosis hormone given out by this gland; JH, the juvenile hormone produced by the corpus allatum.

Both the moulting of the larva and its metamorphosis to the adult are controlled by hormones (Reviews: Seidel 19524, Wigglesworth 1954, Bodenstein 1954). There are at least two main hormones involved, and probably more. The anatomical structures in which the hormones are produced are not always casy to homologise from one group of insects to another, so that the details of the story are complex; only a general summary can be given here. 138 PRINCIPLES OF EMBRYOLOGY

The two hormones which are most fully authenticated are, firstly, a ‘growth and moulting hormone’, which has the effects suggested by its name, and secondly a ‘juvenile’ hormone. The effect of the latter is to prevent the moulting larva from developing into the pupa or adult; metamorphosis is inhibited until the concentration of juvenile hormone, which falls throughout larval life, has sunk low enough. The growth and moulting hormone seems always to be secreted by a gland located in the thoracic region, usually known as the prothoracic gland. The activity of this gland is, however, itself stimulated by an ‘activating’ substance. This is, in many cases, formed by certain neurosecretory cells in the brain; it is sometimes transmitted along the nerves, and in particular into an annexe of the brain known as the corpus cardiacum, from which it may be released into the haemolymph to reach eventually the prothoracic gland. The juvenile hormone is secreted from another organ, known as the corpus allatum; and this again may be activated by influences from the brain, which in this case are probably nervous in nature.

In the higher Diptera, such as Drosophila, the interactions between these organs are made more confusing by the fact that they all lie very close together. The main hormones are produced in an organ known as Weissman’s ring, or the ring gland. This is closely attached to the upper part of the brain, that is to the region of the neurosecretory cells. The ring gland itself is complex; the part nearest the brain corresponds to the corpus cardiacum, that furthest away to the corpus allatum, while the lateral parts probably function as the prothoracic gland (Fig. 8.11).

The growth and moulting hormone is produced periodically towards the end of each instar throughout the whole of larval life. If the source of the juvenile hormone is removed from a young larva (e.g. by extirpating the corpus allatum) a premature metamorphosis occurs, giving rise to a dwarf pupa or adult (Fig. 8.12). On the other hand, if corpora allata from young larvae are transplanted into a larva ready to metamorphose, it is caused to undergo an extra larval moult instead and only finally metamorphoses a stage later, forming a giant. Moreover, by removing the source of metamorphosis hormone when it has begun but not completed its secretion, or by implanting corpora allata from earlier stages, it has been possible to obtain abnormal balances between the two hormones and thus to provoke partial metamorphoses, which produce hemipteran individuals intermediate between nymph and adult, and lepidopterans intermediate between larva and pupa.

In some insects, the life-cycle includes not only moulting and metamorphosis, but also a period of complete standstill, a so-called diapause. It is often in the form of such a resting stage that the animal passes the winter. The diapause may occur either during embryonic development in the egg, or during the early part of the pupal period. The peculiar physiological conditions which enable the animal to survive in a state of arrest have aroused a good deal of interest, and a fair amount has been discovered about them in certain cases. In the Cecropia silkworm (Lepidoptera) Williams (1951) has shown that the diapause, which occurs in early pupal life, is under hormone control. It does not seem to be quite clear how the diapause is initiated; but once the animal has passed into diapause, it remains in that condition until the brain has been cooled for a certain length of time, and then re-warmed. After this alternation of temperatures, the brain is able to emit a hormone which activates the prothoracic gland, and this in its turn produces a second hormone which starts off the development of the pupa into the adult. The effect of the prothoracic hormone can be analysed to some extent in biochemical terms; it causes profound changes in the cytochrome enzymes which are concerned with respiration in the pupal tissues. This brings about a complete change in the metabolic system of the insect, and it is clearly as a result of this change that the development of the various organs is once more able to proceed.

Ficure 8.12

Figures a and b are dwarf pupae of the Wax moth, resulting from the removal of the corpus allatum (source of the moulting hormone) from third and fourth instar larvae; c is a normal pupa, and da giant one produced by implanting an extra corpus allatum from a young larva into one which had already reached the stage at which it would normally pupate. (After Piepho 1943.) e, a precocious adult of the bug Rhodnius produced by joining a Ist-stage larva to a larve undergoing the final moult; f is a normal and-stage larva for comparison. (From Wigglesworth 1934.)

It is perhaps worth emphasising the obvious fact that the metamorphosis and diapause hormones do not determine what type of development any particular tissue will undergo, since they affect equally all the different rudiments. They act as what have been called ‘realisers’, which make it possible for potentialities to become actual, but they are not ‘determiners’, which could change the characters of the reacting tissues. The change they bring about is, at least in the Hemimetabola, to be compared with a modulation (p. 14) rather than a determination. It can be to some extent reversed, since if an adult bug is provided with large amounts of juvenile hormone, it may moult again and the larval characters reappear (Wigglesworth 1948a, b).

4. The determination of imaginal characters

In insects with complete metamorphosis, such as the Diptera, the future imaginal tissue is present during larval life in the form of separate pockets of cells, the so-called imaginal buds. These originate from the hypodermis of the embryo, which is part of the ectoderm. In the early stages of larval life, there is some variation in the readiness with which the different buds react to a given concentration of metamorphosis hormone, and it appears that they undergo, at slightly different rates for different buds, a process of maturation by which they acquire an increasing competence to respond (Bodenstein 1943, 1950). There has been considerable debate as to when these buds become determined in their developmental fate. By irradiating Drosophila embryos with ultraviolet, Geigy (1931) was able to produce purely imaginal defects in animals whose larvae had developed perfectly normally. This occurred only when the irradiation was given about seven hours or more after laying, at a time when the embryo is well on the way to formation. It therefore appears that, long after the period of embryonic determination (which is extremely precocious in Diptera) there is a second period when the imaginal buds are determined. Liischer (1944) has recently produced somewhat similar evidence concerning the Lepidoptera. Evidence tending in the same direction has been brought forward by Gloor (1947) who found that by ether treatment of the young Drosophila embryo he could cause the metathoracic imaginal bud to develop in the way characteristicof the mesothoracic one, a result similar to that produced by the gene bithorax.

It was for some time thought that the determination of the imaginal buds which occurs in mid-embryonic life was the final step which fixed the fate of each part, converting the buds into a rigid mosaic. For instance Bodenstein (1941) found no signs of regulation when limb-buds from third instar larvae were halved. However, Waddington (1942a, b) showed that the determination is by no means final even in larval stages (Fig. 8.13). If early third instar larvae are given a heavy dose of x-rays, many of the imaginal bud cells are killed, and those that remain may produce duplicated organs, or even something quite foreign to their normal fate (e.g. eyes in place of antennae or vice versa). Various authors then found that when the imaginal buds of the larva are cut into fragments which are allowed to develop in isolation, their behaviour is not strictly mosaic. Hadorn and Gloor (1946) showed that the female genital bud behaved like a series of overlapping fields (Fig. 8.14), and Hadorn, Bertani and Gallera (1946) found that in the genital bud of the male considerable reorganisation and regulation of these fields is possible. Vogt (1946) obtained very similar results with the eye-antennal bud. The same author (1946b) studied the development of eye-antennal buds of flies homozygous for the gene aristopedia. This causes part of the antennal bud to develop into a leg instead of an antenna; and it was found that the amount of the bud which is diverted into this abnormal channel of differentiation can be altered by the temperature to which it is subjected during the third larval instar. One must conclude that the determination of the imaginal character is still very labile until at least the time of pupation. Shatoury (1955) argues, on the basis of aberrant types of development found in certain mutant stocks, that the essential features of the imaginal buds are determined by influences from the mesoderm which migrates into the buds during the third instar. Thus the determination of the buds which occurs in the embryo can only be of a preliminary and tentative kind.


Regulative development in Drosophila. On left, a vestigial fly in which one mesothoracic bud has failed to evert and the other produced considerably more than half a thorax. (From Waddington 1953.) On right, conversion of eyes into palps following x-raying of the late larva. (From Waddington 1942b.)


The female genital bud of Drosophila: a shows the genital organs of the adult; Ov, oviduct, at the top the two branches which continue to the ovaries have been cut through; R.S. receptaculum seminalis; Spt., spermatheca; Ut., uterus; Vg., vaginal plate; A.P., anal plates. The larval bud, lying across the intestine, is shown in b, and its position in the larva in c. The results of transplantations of fragments of the bud are summarised in d, which shows the overlapping fields from which the various organs are formed (shading corresponding to those of Figure a). (After Hadorn and Gloor 1946.)

Even during the period of pupation, some degree of regulation is possible to the various imaginal buds. Waddington (1953) and Pantelouris and Waddington (1955) found that if one of the mesothoracic buds is removed, or if it fails to evert, the bud on the other side may regulate so as to form more than the half-thorax which is its normal fate.

Non-mosaic behaviour of a rather different kind is also exhibited by the gonads and genital ducts during the pupal period. Dobzhansky (1931) first pointed out that if the testes of Drosophila simulans, which are normally spiral in shape, fail for some reason to make contact with the ducts which develop from the genital disc, then the spiralisation does not occur, and the testes remain ovoid. Stern (1941) studied the matter in detail, and showed clearly that the ducts induce in the testes the asymmetric growth which leads to the assumption of a spiral shape. In some species of Drosophila, the testes normally grow more or less equally in all directions, and thus remain ovoid; and Stern found that if the larval testis of a species which should develop a spiral gonad becomes attached to the genital duct of a non-spiralising form, then it also fails to become spiral. This is one of the rather few cases in which a species difference is brought about by a difference in an inductive action rather than by being dependent on the nature of the competence of the reacting material. But, as Stern points out, we are dealing here with the transmission of an asymmetric growth stimulus and not with the evocation of histological type of tissue (Fig. 8.15).


Interaction of gonads and genital ducts in Drosophila melanogaster pupae: a shows the outline of the testis at the time it becomes attached to the male duct; 6, the normal coiled form it assumes; c, the uncoiled form resulting from failure of attachment. (From Stern 1941.) Figure d shows the adult female genital tract in a fly from which one larval ovary was removed; the oviduct (arrow) to which no ovary becomes attached fails to elongate. (From Pantelouris 1955.) 144 PRINCIPLES OF EMBRYOLOGY

The reaction between the testis and the ducts is not all in one direction, since pigmented cells migrate out from the testis sheath on to the duct, whose colouration is thus dependent on the kind of testis with which they come in contact (Stern and Hadorn 1939). A more drastic effect of the gonad on the associated duct occurs in females, where Pantelouris (1955) has shown that the lateral branch of the oviduct does not elongate unless it makes contact with the ovary. The ovary itself seems to be relatively independent in its growth, and can, though rather rarely, attain its normal size even when not attached to any genital duct; this may occur even in a male host.

Suggested Reading

Most of the original literature on experiments on embryos is in German; Seidel 1936, 1952a recommended; Haget 1953 is a fine original paper (in French); Wigglesworth 1947 summarises some of the literature; see Poulson 1945 for chromosomal control.

For pupal stages, Hadorn 1948b, Waddington 1942a.

For pupation hormones, Williams 1951, Wigglesworth 1954, Scidel 1952a.

   Principles of Embryology (1956): Part 1 - 1 The Science of Embryology | 2 The Gametes | 3 Fertilisation | 4 Cleavage | 5 The Echinoderms | 6 Spirally Cleaving Eggs | 7 The Ascidians and Amphioxus | 8 The Insects | 9 The Vertebrates: The Amphibia and Birds | 10 The Epigenetics of the Embryonic Axis | 11 Embryo Formation in Other Groups of Vertebrates | 12 Organ Development in Vertebrates | 13 Growth | 14 Regeneration | 15 The Role of Genes in the Epigenetic System | 16 The Activation of Genes by the Cytoplasm | 17 The Synthesis of New Substances | 18 Plasmagenes | 19 The Differentiating System | 20 Individuation - The Formation of Pattern and Shape | References
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