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Corpus allatum activity associated with development of wingbuds in cabbage aphid embryos and larvae
J. Insect Physiol., 1971,Vol. 17,pp. 761 to 773. Pergamon Press. Printed in Great Britain
CORPUS ALLATUM ACTIVITY ASSOCIATED WITH DEVELOPMENT OF WINGBUDS IN CABBAGE APHID EMBRYOS AND LARVAE DINAH
F. WHITE
School of Biological Sciences, Macquarie University, North Ryde, N.S.W. 2113, Australia (Received 6 July 1970) Abstract-Histological studies of cabbage aphid embryos show that the presence or absence of wingbuds and the size of the wingbuds depends on the age and form of the parent aphid. The processes of growth of the wingbuds in alatiform larvae, and their regression in apteriform larvae, are described. The regression of wingbuds bears a close relationship to the activity of the corpus allatum of parents and larvae. Even when prenatally determined as apterae, cabbage aphids seldom lose all trace of wingbuds before birth. The extent of loss of wingbuds in embryos appears to depend on maternal corpus allatum activity and the time during which an embryo is exposed to a particular hormonal environment. In postnatal determination of apterae, the corpus allatum activity of the individual aphid appears to be the controlling factor. It is shown that the results of decapitation experiments carried out by other workers are not necessarily incompatible with this interpretation of form determination. INTRODUCTION THE PHYSIOLOGICAL mechanisms
underlying the control of wing dimorphism in aphids are still a subject of controversy. The problem has resisted solution by any direct approach, because of the small size of the insects and the difficulty of performing suitable surgery on them. Some information, however, can be obtained by histological studies of the developmental processes involved in the production of the different forms. This approach was used by JOHNSONand BIRKS (1960) in a valuable contribution to the theory of control of aphid polymorphism, but further work in more recent years (JOHNSON,1966b ; LEES, 1967) has thrown doubt on the interpretation first offered by Johnson and Birks. In this paper a further examination is made of the developmental process underlying the production of the different forms, in an attempt to clarify the physiological mechanism involved in the determination and differentiation of the two forms. The normal development of the wings is described first. MATERIALS
AND METHODS
The cabbage aphid, Brevicoryne bmssi~ae (L.) was reared on cabbages, as described by LAMBand WHITE (1966). Various procedures used in the preparation of aphids of different ages for histological examination are outlined below. 761
762
DINAH F. WHITE
Embryos Newly ecdysed adult alate and apterous aphids were collected from culture plants and were caged on chemically defined diets (DADD and MITTLER, 1965) in standardized conditions (20°C 70% r.h., and 14 hr photoperiod). Sufficient exercise (either walking or flying) is necessary before alate aphids will settle (JOHNSON, 1954), and denial of exercise may lead to physiological abnormalities in locusts (HIGHNAM and HASKELL, 1964). In the conditions of the present experiments alate aphids began to feed within 48 hr of ecdysis and to reproduce within 72 hr of ecdysis. Except for newly ecdysed alate adults all the slates used were specimens which had commenced feeding, and they were therefore considered to be physiologically ‘normal’. In the same conditions adult apterous aphids were usually active for the first 24 hr after being placed on chemically defined diet but commenced feeding and reproducing within 48 hr. At known times after ecdysis the adult aphids were fixed in Helly’s solution, sectioned serially in the frontal plane at 4 p, and stained with Haidenhain’s azan. The maximum thickness of the mesothoracic wingbud was measured in as many embryos as possible in these adult aphids. The total length of the embryos was also measured as an index of their maturity. Corpus allatum (CA) nuclei were measured in some of the large embryos in parents of both forms and were compared using techniques described elsewhere (WHITE, 1968). First and second instar larvae Presumptive apterous and alate larvae were obtained and treated histologically as described by WHITE (1968). It was known that all larvae reared singly would develop as apterae, whereas of larvae reared in groups, between 50 and 70 per cent would usually become alate. No conditions are known which ensure the exclusive production of alatae in the cabbage aphid and presumptive alatae and apterae are not distinguishable by external features until the third instar.
Third and fourth instar larvae Newly ecdysed third and fourth instar alatiform larvae were collected from culture plants and maintained on chemically defined diet as above. Third instar larvae were fixed, at known times after ecdysis, in alcoholic Bouin’s solution containing O-5 per cent trichloracetic acid and were stained with celestin blue and Mayer’s haemalum, as were the first and second instar larvae. The fourth instar larvae were fixed in Helly’s solution and stained with azan. In all of this work, alate and apterous forms of the same instar were treated in the same way and at the same time and are thus directly comparable. The results for different larval stages (except the first and second stage) were obtained at different times and are therefore However this does not invalidate an account of not quantitatively comparable. wingbud development from the various preparations described above.
DEVELOPMENT
OFWINGBUDS
IN CABBAGE
APHID
EMBRYOSANDLARVAE
763
RESULTS
Chronology of wingbud development The basic features of early wingbud differentiation in aphids have been described (SHULL, 1938; KITZMILLER, 1950; JOHNSON and BIRKS, 1960). However, observations on the cabbage aphid have revealed some differences, particularly in the time-scale of differentiation. These are important in an interpretation of the mechanism of determination of an individual aphid as winged or wingless. Wingbuds are usually recognizable in cabbage aphid embryos before birth. The wingbud takes the form of an area of thickened epidermis, in which the hypodermal cells are elongated or columnar. The wingbud cells are readily distinguished from the more cuboidal or flattened cells of the rest of the epidermis. As there is only a single layer of cells in the wingbud, the thickness of the wingbud is equivalent to the maximum length of the cells. This single-layered stage (Stage I of wingbud development, Fig. la, b) persists through most of the first larval instar, but the thickness of the wingbud increases because of further differentiation and elongation of the cells (see Table 1).
a
b
C
d
FIG. 1. Stages of wingbud development in the cabbage aphid. (a) Wingbuds of a well-developed embryo in an apterous adult (Stage I). (b) Wingbuds of a newborn larva (Stage I). (c) Wingbuds of a late second instar larva. There is an outer layer of orientated cells and a mass of inner cells (Stage II). This specimen shows a late Stage II in which orientation of the inner cells into layers is commencing. (d) Newly ecdysed third instar larva, with wingbuds in the form of flaps (Stage III). (a), (b), (c), (d) are to the same scale. Scale line represents 100 p; for further description see text. c, cuticle; ms, epidermal cells of mesothoracic wingbud; mt, epidermal cells of metathoracic wingbud.
DINAH F. WHITE
764
Following this, there is a re-arrangement of cells accompanied by cell division, so that a structure is formed in which there is an outer layer of cells orientated perpendicular to the body surface and an inner mass (several cells thick) which at first has no obvious cell orientation when seen in frontal sections. This is Stage II of wingbud development (Fig. lc). No attempt has been made to investigate the process by which the inner mass of cells is formed but it is noticeable that there is TABLE ~-THICKNESS OF MESOTHORACIC WINGBUDS DURINGFIRSTANDSECONDLARVAL STAGESIN Brevicwyne brassicae Mean thickness of mesothoracic wingbud Q Larvae of apterous parents
Instar First
Second
Isolated larvae (presumptive apterae)
Grouped larvae (presumptive alatae and apterae)
Larvae of alate parents
0 30-40 hr 48 hrt
14.6 -
14.6 16.7 -
11.7 4.2* -
0 48 hr
4.0 0
23.6
7.0
24-O
0
Time from ecdysis
10.5
* This value may be atypically low as it contained several larvae in which wingbuds were lacking. t 48 hr was chosen for this group rather than 30 to 40 hr because of the slower development of isolated larvae.
no break between mesothoracic and metathoracic wingbuds in the continuity of the original layer of elongated epidermal cells. (Fig. lc) Stage II may begin late in the first instar or early in the second and persists usually throughout the first two days of the second instar (each instar occupied 2-3 days in the conditions of the experiments, except that the fourth larval instar occupied about 5 days in alatiform aphids). During Stage II a further two orientated layers of cells are formed. One of these layers forms the inner layer of cells of the wingbud (i.e. the layer producing the cuticle of the future ventral surface of the adult wing). The other layer of cells becomes the epidermis of the body wall median to the wingbud. In Stage III of their development the wingbuds exist in the form of flaps free from the general epidermal surface except at their anterior ends (Fig. Id). This stage is usually reached late in the second instar although it appears to be quite variable and may be reached early in the instar. According to SHULL (1938) the wingbuds of Macrosiphum solanifolii are ‘distinct sacs’ of this kind during the second instar, although his term ‘evagination’ is perhaps misleading in view of the present observations on the formation of the inner layers of cells.
DEVELOPMENT
OF WINGBUDS
IN CABBAGE APHID EMBRYOS AND LARVAE
765
Further growth and differentiation of the wingbuds occurs in the third larval instar during which the flap-like structure increases in length. At the beginning of the fourth instar, the wingbud occupies the full length of the external cuticular wingpad now present. Soon after ecdysis to the fourth instar, cell division commences in the wingbud epidermis and the wingbud again increases in length. The wingbud becomes secondarily folded within the cuticular wingpad. Secondary folding is evident 48 hr after ecdysis and increases rapidly until the full size of the wing is attained at the end of the instar, when the whole cuticular wingpad is filled with complex folds of wing tissue. The differentiation and growth of the wing rudiments is thus a continuous process. Wingbud development in relation to subsequent alate or apterous form
It is known that the development of cabbage aphids as the apterous form can be determined prenatally by certain factors. Of these factors, the most potent are the form of the parent (alate adults produce almost invariably apterous young) and isolation of the parent (isolated parents produce only apterous young). Other environmental factors such as temperature or nutrition have a less clear-cut effect. Cabbage aphids can also be caused to develop as apterae by certain postnatal conditions; once again, isolation has the strongest effect, and cabbage aphids isolated from birth develop as apterae (WHITE, 1968). The larvae are apparently sensitive to isolation during the 48 hr after birth (-wADA, 1965) and isolation thereafter has no influence on form determination. The period during which determination of the apterous form can occur thus extends from an unknown time before birth to about 48 hr after birth. Wingbuds in embryos related to form and age of parents. In newly ecdysed alate adults, the largest embryo was about 430 p long but most embryos were less than 350 p long. In reproducing alates, the largest embryos were about 500 ~1long. In other words, most of the largest embryos in newly ecdysed adults were about 70 per cent of their expected size at birth, although their growth in the 3 days between the ecdysis of the parent and the beginning of parturition may not be continuous (JOHNSON, 1957). The corresponding sizes of the largest embryos in apterous parents were 400 p at the adult ecdysis and 600 p in reproducing aphids. It is usual in aphids that the young of alate parents are smaller at birth than those of apterous parents. Wingbuds were present, in the form of epidermal thickenings (as described by SHULL, 1938), in most of the large embryos within newly ecdysed alate adults. The relationship between the thickness of the wingbuds and the size of the embryos in alate parents of different ages is shown in Fig. 2. The wmgbuds were nearly always detectable in embryos which were suitably orientated in the sections, but it was not possible to say that every embryo within a newly ecdysed alate parent had wingbuds. Equally, it could not be said that wingbuds were definitely absent in any embryos within a newly ecdysed alate parent. However in alate parents 96 hr after ecdysis (i.e. during the first few days of reproduction) only one embryo had wingbuds distinguishable from the rest of the epidermis, which itself was about 4.2 ,.L
DINAH
766
F. WHITE
thick. This embryo was relatively immature (325 ,Along). Wingbuds were absent in all the larger embryos which were orientated suitably for examination. The situation was different again in older alate parents (10-15 days after ecdysis). In these, the embryos often had large wingbuds, up to 17.6 p thick. Despite this, none of the young reared from these parents became alate.
0
1
I
100
200
LENGTH
I
300
400
500
600
OF EMBRYO L” )
FIG. 2. Thickness of wingbuds in embryos of various sizes in alate parents. Squares: embryos in newly ecdysed alate parents. Black circles: embryos in alate parents 96 hr from ecdysis. Open circles: embryos in alate parents lo-15 days from ecdysis. In embryos in newly ecdysed parents, every measurement represents wingbuds recognizable from the rest of the hypodermis. However in embryos in older parents, the general hypodermis was approximately 4 p thick, so the majority of points on the graph indicate that no wingbuds were recognizable.
DEVELOPMENT
OF WINGBUDS
IN CABBAGE APHID EMBRYOS AND LARVAE
767
Newly ecdysed apterae, in contrast to alates of the same age, contained embryos more than half of which had relatively large wingbuds. Wingbuds were recognizable in most of the remaining embryos, but were small (Fig. 3). The proportion of embryos with large wingbuds increased in older apterae, but the sizes of the wingbuds were not divisible into two distinct groups corresponding to presumptive alatae and presumptive apterae (cf. SHULL, 1938). The thickness of the wingbuds in these embryos ranged from 7 ~1to 22 CL,while in other embryos, wingbuds were absent. Corpus allatum nuclei were measured in four embryos in alate parents and eight embryos in apterous parents. It was usually possible to measure only one embryo in each parent. t-Tests showed that the nuclei were significantly smaller (mean maximum diameter 3.3 CL)in embryos within alate parents than in embryos within apterous parents (mean maximum diameter 4.3 CL,P-C 0.001). First and second instar larvae. At birth, most larvae born to alate parents of unknown ages (i.e. alates simply collected from culture plants and allowed to reproduce) had wingbuds. The thickness of the wingbuds diminished gradually and the wingbuds were no longer distinguishable by the middle of the second instar. Loss of wingbuds followed a similar course in the larvae of apterous parents, when the larvae were determined as apterae by postnatal isolation; in these larvae the wingbuds were larger at birth than the wingbuds in the young of alates, and the initial regression of the wingbuds was also slower (Table 1). Histological examination of grouped young of apterous parents showed wingbuds in various stages of development. In some larvae, the wingbuds were small, as in presumptive apterae. In most, however, the wingbuds were Iarge and it was from these that the stages of growth in the previous section were described. Table 1 summarizes the growth or regression of the wingbuds in the first two instars. The measurements are based on 4 to 10 larvae in each group. On the basis of the size and histological appearance of the wingbuds, it was usually possible to tell whether a second instar cabbage aphid would develop as an aptera or as an alata. It was possible to distinguish some first instar aphids as presumptive apterae, but generally speaking, first instar larvae did not fall into two distinct groups. This is hardly surprising in view of the fact that determination of form may not occur until quite late in the first instar. In prenatal determination of apterae the loss of wingbuds always takes place before Stage II of wingbud development and seems to involve simply the dedifferentiation of the elongated epidermal cells to become normal epidermal cells. In postnatal determination, however, regression of the wingbuds often does not commence until the early part of Stage II of wingbud development, when cell proliferation and rearrangement are leading to the production of a second layer of cells. Histolysis of the inner cells appears to occur in these cases. Differences in CA activity in larvae have already been noted (WHITE, 1968). The major points relevant to this paper are summarized here. Isolated young of apterae had more active CA than grouped young during the first instar. Young of alatae had lower activity of the CA than young of apterae. Also, alatiform larvae
768
DINAH F. WHITE
had less active CA than apteriform larvae in the third and fourth instars (WHITE, 1965 and unpublished reklts).
l
i!O -
0
0
18-
l l l
16l l l
l 0
O0
0
l
L7
l
O0
8 0
0 OO
0 0
l l
8l
0 6-
4-
O 4t
20 I
100
I
200
I
8
300
LENGTH
400
OF EMBRYO
I
I
600
600
$b)
FIG. 3. Thickness of wingbuds in embryos of various sizes in apterous parents.
Squares: embryos in newly ecdysed apterous parents. Black circles: embryos in apterous parents 96 hr from ecdysis. Open circles: embryos in apterous parents lo-15 days from ecdysis. Embryos without detectable wingbuds in parents other than newly ecdysed adults are recorded as having wingbud thickness 4~ (the thickness of the general hypodermis).
DEVELOPMENT OF WINGBUDS
IN CABBAGE APHID EMBRYOS AND LARVAE
769
DISCUSSION A theory of the nature of the developmental process leading to the production of alate and apterous aphids was proposed by JOHNSON and BIRKS (1960) who reviewed most of the relevant earlier work. They suggested that all aphids began development as presumptive alatae, and that apterae became irreversibly diverted from the alate course during development. Thus, they suggested, the endocrine system of the individual would originally be ‘set’ to favour the differentiation of alate structures and it could be ‘reset’ by various stimuli so that differentiation of alate structures was inhibited. More recent papers on wing dimorphism by Johnson have been concerned chiefly with environmental factors influencing form determination (food, crowding, temperature, and photoperiod; JOHNSON, 1965, 1966 a, b). The only modification of the original theory was made in the last of these papers, where Johnson suggested that a positive alata-promoting stimulus might be dispatched to the embryos from the head of the mother. The reason for this modification was the fact that decapitation of adult Aphti craccivora caused them to produce apterae. As a result of observations on the effect of parasitization by wasp larvae on the development of the alate form, JOHNSON(1959) had earlier suggested that alate-apterous polymorphism might be controlled by the metamorphosis hormones. This idea had been foreshadowed in a general sense by WIGGLESWORTH (1954) and more specifically for aphids by LAMB(1956) and KENNEDYand STROYAN (1959). From the results of extensive experiments on the vetch aphid Megoura viciae, LEES (1966, 1967) arrived at different conclusions concerning the process of determination, which in Megoura is always prenatal. Lees considered that embryos were ‘neutral’ in their development up to the time of determination, and found no evidence of a unidirectional shunt or diversion of development from one form to the other. LEES (1961) also expressed the view that the CA was likely to be involved in form determination, and considered that a maternal regulator might augment the activity of the CA of the embryos, leading to production of apterae. More recently LEES (1967) has come to assign a more positive r61e to maternal factors promoting the determination of alatae; once again, this is an attempt to explain the results of decapitation experiments. LEES (1967) however suggested that ‘the maternal factor, which evidently comes from the head region, may well govern the intrinsic activity of the corpus allatum in the late embryos, and may perhaps institute a pattern of reduced secretory activity both before and after birth’. The work described in the present paper permits some clarification of the process of determination in the cabbage aphid and the probable r8le of the CA in this process. A fuller treatment of environmental and physiological factors influencing the form in the cabbage aphid will form the subject of a separate paper; discussion in this paper will therefore be limited to conclusions arising from the results above. On the basis of the observations, it seems reasonable to suppose that all cabbage aphids initially have wing rudiments. These are usually recognizable when the embryos have attained 70 per cent of their length at birth. Sometimes the wing 26
770
DINAH F. WHITE
rudiments are easily identified and sometimes it is difficult to identify them definitely, because of irregularities in the thickness of the general epidermis or because the plane of sectioning is not suitable. However there is no doubt that the majority of presumptive apterae have wingbuds which are often recognizable until the second instar. The only conditions in which wingbuds are definitely absent in embryos are the following: (1) Large embryos in alate parents, particularly at the beginning of the reproductive period; and (‘2) Occasionally in embryos at the upper end of the size range in apterous parents. It is suggested that in each of these the prenatal loss of wingbuds may be explained in terms of the CA activity of the mother. High CA activity in alate adults commences soon after the imaginal ecdysis. On the other hand, in apterae, CA activity drops sharply from a high level in the fourth instar to a relatively low level throughout adult life (WHITE, 1965). Th ese results, based on the volume of the CA, have since been confirmed by a further series of preparations in which the activity of the gland was assessed by measurement of the nuclei (White, unpublished). Thus, in young alate parents, the embryos are probably subjected to a high and rapidly increasing concentration of juvenile hormone, which would inhibit the differentiation of ‘adult’ tissues such as wingbuds. It is well established that differentiation of adult tissues may not only be inhibited but also reversed, by continued exposure to juvenile hormone (LAWRENCE,1966). Wingbuds are present in the embryos just after the adult ecdysis, before activity of the CA increases, but the wingbuds are lost during the following 2 to 3 days before the adult begins to reproduce. CA activity remains relatively high in alate adults until senescence; the persistence of wingbuds in embryos in older alate parents may perhaps be partly explained by the fact that in newly ecdysed alate parents very few embryos are developed so that more juvenile hormone might be available to each one. Later there is several times the amount of embryonic tissue to compete for the hormone. However during the investigation, the young of alates were almost always apterae whether or not their wingbuds were lost before birth. The observations on embryos in young alates suggest that embryos can be irreversibly affected by maternal hormones in the 2 to 3 days before birth. The presence of wingbuds in embryos in young apterous adults suggests further that the development of the wing rudiments up to a certain stage may be independent of maternal juvenile hormone concentration, because they develop despite high CA activity during the fourth instar of the apterous parents. This again emphasizes the importance of the last few days of embryonic life in prenatal form determination in the cabbage aphid. It has been shown that environmental conditions can modify CA activity in adult apterae (WHITE, 1968 and unpublished results) and such conditions may cause regression of wingbuds to commence in the embryos. Wingbuds are rarely found to be completely lacking in embryos in apterous parents. When they are lacking, it is frequently in embryos which are larger than average, suggesting that they may have been exposed for an abnormally long time to the maternal hormonal environment, because of a delay in parturition. Although the number of embryos
DEVELOPMENT
OF WINGBUDS
IN CABBAGE APHID EMBRYOS AND LARVAE
771
without wingbuds was small and any suggestion about a relationship between size and wingbud loss is only tentative, it emphasizes the importance of a consideration of a ‘time factor’ in the process of determination. The length of time during which an embryo or young larva is exposed to a stimulus or more specifically to a hormonal environment is important, just as the strength of the stimulus (or concentration of the hormone) and the stage of development at which the stimulus is applied are important. There is no evidence in the present work to support the view (LEES, 1961,1967) that the first step in prenatal determination of apterae is the activation of the embryonic CA by a maternal factor. The CA in embryos in alate parents (i.e. embryos which were presumptive apterae) were less active than in embryos in apterous parents (i.e. embryos of which a majority would develop as alates). Even after birth the young of alates had less active CA. Therefore prenatal form determination in the young of alates seems more likely to be caused entirely by maternal juvenile hormone than by the action of an unknown factor influencing the CA activity of the embryos themselves. This is not to say that the CA of an individual aphid cannot be involved in the determination of form of that individual. The postnatal determination of apterae by isolation in the first instar is clearly associated with increased CA activity (WHITE, 1968) and one might expect also that certain extrinsic factors might also affect CA activity in embryos directly. Temperature could perhaps be one such factor. Observations on the cabbage aphid favour a positive role for environmental and physiological stimuli which result in the determination of apterae. This is in accordance with the view that the alate form is the more primitive phylogenetically, and with the observation of wing rudiments in embryos which are presumptive apterae. LEES (1966) has commented that wing rudiments of aphid embryos may have no greater significance than the gill pouches of an embryonic mammal. This analogy is perhaps not entirely valid, because the wing rudiments are in fact the precursors of wings in the adult and as long as the wing rudiments continue to grow, the aphid develops as an alata. LEES (1966) has suggested further that the embryonic aphid should be considered merely as a ‘virginopara’ rather than as an incipient alata or aptera and has argued that the results of decapitation experiments provide evidence against determination of form resulting from diversion towards winglessness. Although this view incorporates the fact that the embryo originally has the potential to develop as an alata or as an aptera, it implies the existence of two separate physiological ‘switches’, one to direct the development of alates and one to direct the development of apterae. This complication does not seem necessary at the present time to a theory of alternative dimorphism. A more serious objection is that unidirectional diversion to the apterous pathway of development does occur; once an embryo or young larval aphid has lost its wing rudiments it has lost the potential to develop as an alate. On the other hand, until the wing rudiments have reached a certain stage of development (the beginning of Stage II ?) they can still respond to some environmental and physiological stimuli by regressing.
772
DINAH F. WHITE
We are thus left essentially with the theory presented by JOHNSON and BIRKS (1960). The only reason for modifying this theory was the fact that decapitated aphids (A. craccivora and ikl. ericiae) produce only apterous young, whereas if the maternal control of form determination originated in the head of the mother and was a positive stimulus to aptera production, decapitated aphids would be expected to produce alate young. Since the original theory (JOHNSON and BIRKS, 1960) is satisfactory in other respects, it is worth considering whether there are any other possible explanations of the apparent anomaly. A simple alternative explanation would be that severe wounding of the parent might stimulate the CA of the embryos. Severe wounding was found to have an apterizing effect on third and fourth instar alatiform aphid larvae (JOHNSON,1959 ; JOHNSON and BIRKS, 1960) although the same authors did not find that severe wounding of adults caused the production of apterous young. Wounding is known to stimulate CA activity in other insects (NEEDHAM, 1965) but the relationships between regeneration, CA activity and prothoracic gland activity are complex (O’FARRELL et al., 1960). It is quite likely that severe wounding of the parent, without decapitation, might not be sufficient to affect the form of the young. Decapitation, however, is much more drastic than any kind of cutting or burning of the thorax or abdomen, and destroys many organ systems (e.g. nervous system, alimentary canal, and dorsal aorta). In particular it destroys the brain and corpus cardiacum/CA system of the parent. Any stimulus to raised CA activity (e.g. from wounding) can thus be received only by the embryos. It is difficult to test this suggestion satisfactorily, because the young of decapitated parents are born prematurely and the young of wounded parents are born after a delay, and they are therefore not equivalent in size or physiological condition. However, it can be seen from this discussion that it is not necessary to postulate an ‘alata-determiner’ to account for the results of decapitation experiments. Acknowledgements-The author thanks Dr. D. T. timON of Sydney University, and Professor K. P. LAMB of the University of Papua and New Guinea, for their advice during the course of this work, and for their helpful criticism of the manuscript. Most of the work described was carried out in the School of Biological Sciences, Sydney University, during the tenure of a C.S.I.R.O. Postgraduate Studentship. REFERENCES DADDR. H. and MITTLER T. E. (1965) Studies on the artificial feeding of the aphid Myers persicue (Sulser)-III. Some major nutritional requirements. J. Insect Physiol. 11, 717-743. HIGHNAMK. C. and HA~KELLP. T. (1964) Th e endocrine systems of isolated and crowded Locusta and Schistocerca in relation to o&zyte growth, and the effects of flying upon maturation. y. Insect Physiol. 10,849-864. JOHNSONB. (1954) Effect of flight on behaviour of Aphisfabae Stop. Nature, Lond. 173,831. JOHNSONB. (1957) Studies on the degeneration of the Ilight muscles of alate aphids-I. A comparative study of the occurrence of muscle breakdown in relation to reproduction in several species. J. Insect Physiol. 1, 248-256. JOHNSONB. (1959) Effect of parasitisation by Aphidius platensis Brethes on the developmental physiology of its host Aphis craccivora Koch. Entomologia exp. appl. 2, 82-99.
DEVELOPMENT OF WINGBUDSIN CABBAGE APHIDEMBRYOSANDLARVAE
773
JOHNSON B. (1965) Wing polymorphism in aphids-H. Interaction between aphids. Entomologia exp. appl. 8, 49-64. JOHNSONB. (1966a) Wing polymorphism in aphids-III. The influence of the host plant. Entomologia exp. appl. 9, 213-222. JOHNSONB. (1966b) Wing polymorphism in aphids-IV. The effect of temperature and photoperiod. Entomologia exp. appl. 9, 301-313. JOHNSONB. and BIRKS P. R. (1960) Studies on wing polymorphism in aphids-I. The developmental process involved in the production of the different forms. Entomologia exp. appl. 3, 327-339. KAWADAK. (1965) The development of winged forms in the cabbage aphid, Brevicoryne brassicae Linnaeus-II. The period of determination of wing development. Ber. Ohara Inst. landw. Biol. 13, l-5. KENNEDYJ. S. and STROYANH. L. G. (1959) Biology of aphids. A. Rev. Ent. 4, 139-160. KITZMILLERJ. B. (1950) The time interval between determination and differentiation of wings, ocelli and wing muscles in the aphid Macrosiphum sanborni (Gillette). Am. Nut. 84, 23-50. LAMB K. P. (1956) Physiological relations between aphids and their host plants. Ph.D. Thesis, University of Cambridge. LAMB K. P. and WHITE D. (1966) Effect of temperature, starvation and crowding on production of alate young by the cabbage aphid (Brevicoryne brassicae). Entomologia exp. appl. 9, 179-184. LA~EENCE P. A. (1966) The hormonal control of the development of hairs and bristles in the milkweed bug, Oncopeltus fasciatus Dall. J. exp. Biol. 44, 507-522. LEES A. D. (1961) Clonal polymorphism in aphids. Symp. R. e-nt. Sot. Lond. 1, 68-79. LEES A. D. (1966) The control of polymorphism in aphids. Adw. Insect Physiol. 3,207-277. LEES A. D. (1967) The production of the apterous and alate forms in the aphid Megoura viciae Buckton, with special reference to the r6le of crowding, J. Insect Physiol. 13, 289-318. NEEDHAMA. E. (1965) In Regeneration in Animals and Related Problems (Ed. by KIORTISV. and TEAMPUSCHH. A. L.). North Holland, Amsterdam. O’FAEEELL A. F., STOCKA., RAE C. A., and MORGANJ. A. (1960) Regeneration and development in the cockroach Blatella germanica. C?as. Est. Spol. ent. 57, 317-324. SHULL A. F. (1938) Time of determination and time of differentiation of aphid wings. Am. Nat. 72, 170-179, WHITE D. (1965) Changes in size of the corpus allatum of a polymorphic insect. Nature, Lond. 208, 807. WHITE D. F. (1968) Cabbage aphid: effect of isolation on form and on endocrine activity. Science, Wash. 159, 218-219. WIGGLE~WORTH V. B. (1954) The Physiology of Insect Metamorphosis. Cambridge University Press, Cambridge.
CORPUS ALLATUM ACTIVITY ASSOCIATED WITH DEVELOPMENT OF WINGBUDS IN CABBAGE APHID EMBRYOS AND LARVAE DINAH
F. WHITE
School of Biological Sciences, Macquarie University, North Ryde, N.S.W. 2113, Australia (Received 6 July 1970) Abstract-Histological studies of cabbage aphid embryos show that the presence or absence of wingbuds and the size of the wingbuds depends on the age and form of the parent aphid. The processes of growth of the wingbuds in alatiform larvae, and their regression in apteriform larvae, are described. The regression of wingbuds bears a close relationship to the activity of the corpus allatum of parents and larvae. Even when prenatally determined as apterae, cabbage aphids seldom lose all trace of wingbuds before birth. The extent of loss of wingbuds in embryos appears to depend on maternal corpus allatum activity and the time during which an embryo is exposed to a particular hormonal environment. In postnatal determination of apterae, the corpus allatum activity of the individual aphid appears to be the controlling factor. It is shown that the results of decapitation experiments carried out by other workers are not necessarily incompatible with this interpretation of form determination. INTRODUCTION THE PHYSIOLOGICAL mechanisms
underlying the control of wing dimorphism in aphids are still a subject of controversy. The problem has resisted solution by any direct approach, because of the small size of the insects and the difficulty of performing suitable surgery on them. Some information, however, can be obtained by histological studies of the developmental processes involved in the production of the different forms. This approach was used by JOHNSONand BIRKS (1960) in a valuable contribution to the theory of control of aphid polymorphism, but further work in more recent years (JOHNSON,1966b ; LEES, 1967) has thrown doubt on the interpretation first offered by Johnson and Birks. In this paper a further examination is made of the developmental process underlying the production of the different forms, in an attempt to clarify the physiological mechanism involved in the determination and differentiation of the two forms. The normal development of the wings is described first. MATERIALS
AND METHODS
The cabbage aphid, Brevicoryne bmssi~ae (L.) was reared on cabbages, as described by LAMBand WHITE (1966). Various procedures used in the preparation of aphids of different ages for histological examination are outlined below. 761
762
DINAH F. WHITE
Embryos Newly ecdysed adult alate and apterous aphids were collected from culture plants and were caged on chemically defined diets (DADD and MITTLER, 1965) in standardized conditions (20°C 70% r.h., and 14 hr photoperiod). Sufficient exercise (either walking or flying) is necessary before alate aphids will settle (JOHNSON, 1954), and denial of exercise may lead to physiological abnormalities in locusts (HIGHNAM and HASKELL, 1964). In the conditions of the present experiments alate aphids began to feed within 48 hr of ecdysis and to reproduce within 72 hr of ecdysis. Except for newly ecdysed alate adults all the slates used were specimens which had commenced feeding, and they were therefore considered to be physiologically ‘normal’. In the same conditions adult apterous aphids were usually active for the first 24 hr after being placed on chemically defined diet but commenced feeding and reproducing within 48 hr. At known times after ecdysis the adult aphids were fixed in Helly’s solution, sectioned serially in the frontal plane at 4 p, and stained with Haidenhain’s azan. The maximum thickness of the mesothoracic wingbud was measured in as many embryos as possible in these adult aphids. The total length of the embryos was also measured as an index of their maturity. Corpus allatum (CA) nuclei were measured in some of the large embryos in parents of both forms and were compared using techniques described elsewhere (WHITE, 1968). First and second instar larvae Presumptive apterous and alate larvae were obtained and treated histologically as described by WHITE (1968). It was known that all larvae reared singly would develop as apterae, whereas of larvae reared in groups, between 50 and 70 per cent would usually become alate. No conditions are known which ensure the exclusive production of alatae in the cabbage aphid and presumptive alatae and apterae are not distinguishable by external features until the third instar.
Third and fourth instar larvae Newly ecdysed third and fourth instar alatiform larvae were collected from culture plants and maintained on chemically defined diet as above. Third instar larvae were fixed, at known times after ecdysis, in alcoholic Bouin’s solution containing O-5 per cent trichloracetic acid and were stained with celestin blue and Mayer’s haemalum, as were the first and second instar larvae. The fourth instar larvae were fixed in Helly’s solution and stained with azan. In all of this work, alate and apterous forms of the same instar were treated in the same way and at the same time and are thus directly comparable. The results for different larval stages (except the first and second stage) were obtained at different times and are therefore However this does not invalidate an account of not quantitatively comparable. wingbud development from the various preparations described above.
DEVELOPMENT
OFWINGBUDS
IN CABBAGE
APHID
EMBRYOSANDLARVAE
763
RESULTS
Chronology of wingbud development The basic features of early wingbud differentiation in aphids have been described (SHULL, 1938; KITZMILLER, 1950; JOHNSON and BIRKS, 1960). However, observations on the cabbage aphid have revealed some differences, particularly in the time-scale of differentiation. These are important in an interpretation of the mechanism of determination of an individual aphid as winged or wingless. Wingbuds are usually recognizable in cabbage aphid embryos before birth. The wingbud takes the form of an area of thickened epidermis, in which the hypodermal cells are elongated or columnar. The wingbud cells are readily distinguished from the more cuboidal or flattened cells of the rest of the epidermis. As there is only a single layer of cells in the wingbud, the thickness of the wingbud is equivalent to the maximum length of the cells. This single-layered stage (Stage I of wingbud development, Fig. la, b) persists through most of the first larval instar, but the thickness of the wingbud increases because of further differentiation and elongation of the cells (see Table 1).
a
b
C
d
FIG. 1. Stages of wingbud development in the cabbage aphid. (a) Wingbuds of a well-developed embryo in an apterous adult (Stage I). (b) Wingbuds of a newborn larva (Stage I). (c) Wingbuds of a late second instar larva. There is an outer layer of orientated cells and a mass of inner cells (Stage II). This specimen shows a late Stage II in which orientation of the inner cells into layers is commencing. (d) Newly ecdysed third instar larva, with wingbuds in the form of flaps (Stage III). (a), (b), (c), (d) are to the same scale. Scale line represents 100 p; for further description see text. c, cuticle; ms, epidermal cells of mesothoracic wingbud; mt, epidermal cells of metathoracic wingbud.
DINAH F. WHITE
764
Following this, there is a re-arrangement of cells accompanied by cell division, so that a structure is formed in which there is an outer layer of cells orientated perpendicular to the body surface and an inner mass (several cells thick) which at first has no obvious cell orientation when seen in frontal sections. This is Stage II of wingbud development (Fig. lc). No attempt has been made to investigate the process by which the inner mass of cells is formed but it is noticeable that there is TABLE ~-THICKNESS OF MESOTHORACIC WINGBUDS DURINGFIRSTANDSECONDLARVAL STAGESIN Brevicwyne brassicae Mean thickness of mesothoracic wingbud Q Larvae of apterous parents
Instar First
Second
Isolated larvae (presumptive apterae)
Grouped larvae (presumptive alatae and apterae)
Larvae of alate parents
0 30-40 hr 48 hrt
14.6 -
14.6 16.7 -
11.7 4.2* -
0 48 hr
4.0 0
23.6
7.0
24-O
0
Time from ecdysis
10.5
* This value may be atypically low as it contained several larvae in which wingbuds were lacking. t 48 hr was chosen for this group rather than 30 to 40 hr because of the slower development of isolated larvae.
no break between mesothoracic and metathoracic wingbuds in the continuity of the original layer of elongated epidermal cells. (Fig. lc) Stage II may begin late in the first instar or early in the second and persists usually throughout the first two days of the second instar (each instar occupied 2-3 days in the conditions of the experiments, except that the fourth larval instar occupied about 5 days in alatiform aphids). During Stage II a further two orientated layers of cells are formed. One of these layers forms the inner layer of cells of the wingbud (i.e. the layer producing the cuticle of the future ventral surface of the adult wing). The other layer of cells becomes the epidermis of the body wall median to the wingbud. In Stage III of their development the wingbuds exist in the form of flaps free from the general epidermal surface except at their anterior ends (Fig. Id). This stage is usually reached late in the second instar although it appears to be quite variable and may be reached early in the instar. According to SHULL (1938) the wingbuds of Macrosiphum solanifolii are ‘distinct sacs’ of this kind during the second instar, although his term ‘evagination’ is perhaps misleading in view of the present observations on the formation of the inner layers of cells.
DEVELOPMENT
OF WINGBUDS
IN CABBAGE APHID EMBRYOS AND LARVAE
765
Further growth and differentiation of the wingbuds occurs in the third larval instar during which the flap-like structure increases in length. At the beginning of the fourth instar, the wingbud occupies the full length of the external cuticular wingpad now present. Soon after ecdysis to the fourth instar, cell division commences in the wingbud epidermis and the wingbud again increases in length. The wingbud becomes secondarily folded within the cuticular wingpad. Secondary folding is evident 48 hr after ecdysis and increases rapidly until the full size of the wing is attained at the end of the instar, when the whole cuticular wingpad is filled with complex folds of wing tissue. The differentiation and growth of the wing rudiments is thus a continuous process. Wingbud development in relation to subsequent alate or apterous form
It is known that the development of cabbage aphids as the apterous form can be determined prenatally by certain factors. Of these factors, the most potent are the form of the parent (alate adults produce almost invariably apterous young) and isolation of the parent (isolated parents produce only apterous young). Other environmental factors such as temperature or nutrition have a less clear-cut effect. Cabbage aphids can also be caused to develop as apterae by certain postnatal conditions; once again, isolation has the strongest effect, and cabbage aphids isolated from birth develop as apterae (WHITE, 1968). The larvae are apparently sensitive to isolation during the 48 hr after birth (-wADA, 1965) and isolation thereafter has no influence on form determination. The period during which determination of the apterous form can occur thus extends from an unknown time before birth to about 48 hr after birth. Wingbuds in embryos related to form and age of parents. In newly ecdysed alate adults, the largest embryo was about 430 p long but most embryos were less than 350 p long. In reproducing alates, the largest embryos were about 500 ~1long. In other words, most of the largest embryos in newly ecdysed adults were about 70 per cent of their expected size at birth, although their growth in the 3 days between the ecdysis of the parent and the beginning of parturition may not be continuous (JOHNSON, 1957). The corresponding sizes of the largest embryos in apterous parents were 400 p at the adult ecdysis and 600 p in reproducing aphids. It is usual in aphids that the young of alate parents are smaller at birth than those of apterous parents. Wingbuds were present, in the form of epidermal thickenings (as described by SHULL, 1938), in most of the large embryos within newly ecdysed alate adults. The relationship between the thickness of the wingbuds and the size of the embryos in alate parents of different ages is shown in Fig. 2. The wmgbuds were nearly always detectable in embryos which were suitably orientated in the sections, but it was not possible to say that every embryo within a newly ecdysed alate parent had wingbuds. Equally, it could not be said that wingbuds were definitely absent in any embryos within a newly ecdysed alate parent. However in alate parents 96 hr after ecdysis (i.e. during the first few days of reproduction) only one embryo had wingbuds distinguishable from the rest of the epidermis, which itself was about 4.2 ,.L
DINAH
766
F. WHITE
thick. This embryo was relatively immature (325 ,Along). Wingbuds were absent in all the larger embryos which were orientated suitably for examination. The situation was different again in older alate parents (10-15 days after ecdysis). In these, the embryos often had large wingbuds, up to 17.6 p thick. Despite this, none of the young reared from these parents became alate.
0
1
I
100
200
LENGTH
I
300
400
500
600
OF EMBRYO L” )
FIG. 2. Thickness of wingbuds in embryos of various sizes in alate parents. Squares: embryos in newly ecdysed alate parents. Black circles: embryos in alate parents 96 hr from ecdysis. Open circles: embryos in alate parents lo-15 days from ecdysis. In embryos in newly ecdysed parents, every measurement represents wingbuds recognizable from the rest of the hypodermis. However in embryos in older parents, the general hypodermis was approximately 4 p thick, so the majority of points on the graph indicate that no wingbuds were recognizable.
DEVELOPMENT
OF WINGBUDS
IN CABBAGE APHID EMBRYOS AND LARVAE
767
Newly ecdysed apterae, in contrast to alates of the same age, contained embryos more than half of which had relatively large wingbuds. Wingbuds were recognizable in most of the remaining embryos, but were small (Fig. 3). The proportion of embryos with large wingbuds increased in older apterae, but the sizes of the wingbuds were not divisible into two distinct groups corresponding to presumptive alatae and presumptive apterae (cf. SHULL, 1938). The thickness of the wingbuds in these embryos ranged from 7 ~1to 22 CL,while in other embryos, wingbuds were absent. Corpus allatum nuclei were measured in four embryos in alate parents and eight embryos in apterous parents. It was usually possible to measure only one embryo in each parent. t-Tests showed that the nuclei were significantly smaller (mean maximum diameter 3.3 CL)in embryos within alate parents than in embryos within apterous parents (mean maximum diameter 4.3 CL,P-C 0.001). First and second instar larvae. At birth, most larvae born to alate parents of unknown ages (i.e. alates simply collected from culture plants and allowed to reproduce) had wingbuds. The thickness of the wingbuds diminished gradually and the wingbuds were no longer distinguishable by the middle of the second instar. Loss of wingbuds followed a similar course in the larvae of apterous parents, when the larvae were determined as apterae by postnatal isolation; in these larvae the wingbuds were larger at birth than the wingbuds in the young of alates, and the initial regression of the wingbuds was also slower (Table 1). Histological examination of grouped young of apterous parents showed wingbuds in various stages of development. In some larvae, the wingbuds were small, as in presumptive apterae. In most, however, the wingbuds were Iarge and it was from these that the stages of growth in the previous section were described. Table 1 summarizes the growth or regression of the wingbuds in the first two instars. The measurements are based on 4 to 10 larvae in each group. On the basis of the size and histological appearance of the wingbuds, it was usually possible to tell whether a second instar cabbage aphid would develop as an aptera or as an alata. It was possible to distinguish some first instar aphids as presumptive apterae, but generally speaking, first instar larvae did not fall into two distinct groups. This is hardly surprising in view of the fact that determination of form may not occur until quite late in the first instar. In prenatal determination of apterae the loss of wingbuds always takes place before Stage II of wingbud development and seems to involve simply the dedifferentiation of the elongated epidermal cells to become normal epidermal cells. In postnatal determination, however, regression of the wingbuds often does not commence until the early part of Stage II of wingbud development, when cell proliferation and rearrangement are leading to the production of a second layer of cells. Histolysis of the inner cells appears to occur in these cases. Differences in CA activity in larvae have already been noted (WHITE, 1968). The major points relevant to this paper are summarized here. Isolated young of apterae had more active CA than grouped young during the first instar. Young of alatae had lower activity of the CA than young of apterae. Also, alatiform larvae
768
DINAH F. WHITE
had less active CA than apteriform larvae in the third and fourth instars (WHITE, 1965 and unpublished reklts).
l
i!O -
0
0
18-
l l l
16l l l
l 0
O0
0
l
L7
l
O0
8 0
0 OO
0 0
l l
8l
0 6-
4-
O 4t
20 I
100
I
200
I
8
300
LENGTH
400
OF EMBRYO
I
I
600
600
$b)
FIG. 3. Thickness of wingbuds in embryos of various sizes in apterous parents.
Squares: embryos in newly ecdysed apterous parents. Black circles: embryos in apterous parents 96 hr from ecdysis. Open circles: embryos in apterous parents lo-15 days from ecdysis. Embryos without detectable wingbuds in parents other than newly ecdysed adults are recorded as having wingbud thickness 4~ (the thickness of the general hypodermis).
DEVELOPMENT OF WINGBUDS
IN CABBAGE APHID EMBRYOS AND LARVAE
769
DISCUSSION A theory of the nature of the developmental process leading to the production of alate and apterous aphids was proposed by JOHNSON and BIRKS (1960) who reviewed most of the relevant earlier work. They suggested that all aphids began development as presumptive alatae, and that apterae became irreversibly diverted from the alate course during development. Thus, they suggested, the endocrine system of the individual would originally be ‘set’ to favour the differentiation of alate structures and it could be ‘reset’ by various stimuli so that differentiation of alate structures was inhibited. More recent papers on wing dimorphism by Johnson have been concerned chiefly with environmental factors influencing form determination (food, crowding, temperature, and photoperiod; JOHNSON, 1965, 1966 a, b). The only modification of the original theory was made in the last of these papers, where Johnson suggested that a positive alata-promoting stimulus might be dispatched to the embryos from the head of the mother. The reason for this modification was the fact that decapitation of adult Aphti craccivora caused them to produce apterae. As a result of observations on the effect of parasitization by wasp larvae on the development of the alate form, JOHNSON(1959) had earlier suggested that alate-apterous polymorphism might be controlled by the metamorphosis hormones. This idea had been foreshadowed in a general sense by WIGGLESWORTH (1954) and more specifically for aphids by LAMB(1956) and KENNEDYand STROYAN (1959). From the results of extensive experiments on the vetch aphid Megoura viciae, LEES (1966, 1967) arrived at different conclusions concerning the process of determination, which in Megoura is always prenatal. Lees considered that embryos were ‘neutral’ in their development up to the time of determination, and found no evidence of a unidirectional shunt or diversion of development from one form to the other. LEES (1961) also expressed the view that the CA was likely to be involved in form determination, and considered that a maternal regulator might augment the activity of the CA of the embryos, leading to production of apterae. More recently LEES (1967) has come to assign a more positive r61e to maternal factors promoting the determination of alatae; once again, this is an attempt to explain the results of decapitation experiments. LEES (1967) however suggested that ‘the maternal factor, which evidently comes from the head region, may well govern the intrinsic activity of the corpus allatum in the late embryos, and may perhaps institute a pattern of reduced secretory activity both before and after birth’. The work described in the present paper permits some clarification of the process of determination in the cabbage aphid and the probable r8le of the CA in this process. A fuller treatment of environmental and physiological factors influencing the form in the cabbage aphid will form the subject of a separate paper; discussion in this paper will therefore be limited to conclusions arising from the results above. On the basis of the observations, it seems reasonable to suppose that all cabbage aphids initially have wing rudiments. These are usually recognizable when the embryos have attained 70 per cent of their length at birth. Sometimes the wing 26
770
DINAH F. WHITE
rudiments are easily identified and sometimes it is difficult to identify them definitely, because of irregularities in the thickness of the general epidermis or because the plane of sectioning is not suitable. However there is no doubt that the majority of presumptive apterae have wingbuds which are often recognizable until the second instar. The only conditions in which wingbuds are definitely absent in embryos are the following: (1) Large embryos in alate parents, particularly at the beginning of the reproductive period; and (‘2) Occasionally in embryos at the upper end of the size range in apterous parents. It is suggested that in each of these the prenatal loss of wingbuds may be explained in terms of the CA activity of the mother. High CA activity in alate adults commences soon after the imaginal ecdysis. On the other hand, in apterae, CA activity drops sharply from a high level in the fourth instar to a relatively low level throughout adult life (WHITE, 1965). Th ese results, based on the volume of the CA, have since been confirmed by a further series of preparations in which the activity of the gland was assessed by measurement of the nuclei (White, unpublished). Thus, in young alate parents, the embryos are probably subjected to a high and rapidly increasing concentration of juvenile hormone, which would inhibit the differentiation of ‘adult’ tissues such as wingbuds. It is well established that differentiation of adult tissues may not only be inhibited but also reversed, by continued exposure to juvenile hormone (LAWRENCE,1966). Wingbuds are present in the embryos just after the adult ecdysis, before activity of the CA increases, but the wingbuds are lost during the following 2 to 3 days before the adult begins to reproduce. CA activity remains relatively high in alate adults until senescence; the persistence of wingbuds in embryos in older alate parents may perhaps be partly explained by the fact that in newly ecdysed alate parents very few embryos are developed so that more juvenile hormone might be available to each one. Later there is several times the amount of embryonic tissue to compete for the hormone. However during the investigation, the young of alates were almost always apterae whether or not their wingbuds were lost before birth. The observations on embryos in young alates suggest that embryos can be irreversibly affected by maternal hormones in the 2 to 3 days before birth. The presence of wingbuds in embryos in young apterous adults suggests further that the development of the wing rudiments up to a certain stage may be independent of maternal juvenile hormone concentration, because they develop despite high CA activity during the fourth instar of the apterous parents. This again emphasizes the importance of the last few days of embryonic life in prenatal form determination in the cabbage aphid. It has been shown that environmental conditions can modify CA activity in adult apterae (WHITE, 1968 and unpublished results) and such conditions may cause regression of wingbuds to commence in the embryos. Wingbuds are rarely found to be completely lacking in embryos in apterous parents. When they are lacking, it is frequently in embryos which are larger than average, suggesting that they may have been exposed for an abnormally long time to the maternal hormonal environment, because of a delay in parturition. Although the number of embryos
DEVELOPMENT
OF WINGBUDS
IN CABBAGE APHID EMBRYOS AND LARVAE
771
without wingbuds was small and any suggestion about a relationship between size and wingbud loss is only tentative, it emphasizes the importance of a consideration of a ‘time factor’ in the process of determination. The length of time during which an embryo or young larva is exposed to a stimulus or more specifically to a hormonal environment is important, just as the strength of the stimulus (or concentration of the hormone) and the stage of development at which the stimulus is applied are important. There is no evidence in the present work to support the view (LEES, 1961,1967) that the first step in prenatal determination of apterae is the activation of the embryonic CA by a maternal factor. The CA in embryos in alate parents (i.e. embryos which were presumptive apterae) were less active than in embryos in apterous parents (i.e. embryos of which a majority would develop as alates). Even after birth the young of alates had less active CA. Therefore prenatal form determination in the young of alates seems more likely to be caused entirely by maternal juvenile hormone than by the action of an unknown factor influencing the CA activity of the embryos themselves. This is not to say that the CA of an individual aphid cannot be involved in the determination of form of that individual. The postnatal determination of apterae by isolation in the first instar is clearly associated with increased CA activity (WHITE, 1968) and one might expect also that certain extrinsic factors might also affect CA activity in embryos directly. Temperature could perhaps be one such factor. Observations on the cabbage aphid favour a positive role for environmental and physiological stimuli which result in the determination of apterae. This is in accordance with the view that the alate form is the more primitive phylogenetically, and with the observation of wing rudiments in embryos which are presumptive apterae. LEES (1966) has commented that wing rudiments of aphid embryos may have no greater significance than the gill pouches of an embryonic mammal. This analogy is perhaps not entirely valid, because the wing rudiments are in fact the precursors of wings in the adult and as long as the wing rudiments continue to grow, the aphid develops as an alata. LEES (1966) has suggested further that the embryonic aphid should be considered merely as a ‘virginopara’ rather than as an incipient alata or aptera and has argued that the results of decapitation experiments provide evidence against determination of form resulting from diversion towards winglessness. Although this view incorporates the fact that the embryo originally has the potential to develop as an alata or as an aptera, it implies the existence of two separate physiological ‘switches’, one to direct the development of alates and one to direct the development of apterae. This complication does not seem necessary at the present time to a theory of alternative dimorphism. A more serious objection is that unidirectional diversion to the apterous pathway of development does occur; once an embryo or young larval aphid has lost its wing rudiments it has lost the potential to develop as an alate. On the other hand, until the wing rudiments have reached a certain stage of development (the beginning of Stage II ?) they can still respond to some environmental and physiological stimuli by regressing.
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DINAH F. WHITE
We are thus left essentially with the theory presented by JOHNSON and BIRKS (1960). The only reason for modifying this theory was the fact that decapitated aphids (A. craccivora and ikl. ericiae) produce only apterous young, whereas if the maternal control of form determination originated in the head of the mother and was a positive stimulus to aptera production, decapitated aphids would be expected to produce alate young. Since the original theory (JOHNSON and BIRKS, 1960) is satisfactory in other respects, it is worth considering whether there are any other possible explanations of the apparent anomaly. A simple alternative explanation would be that severe wounding of the parent might stimulate the CA of the embryos. Severe wounding was found to have an apterizing effect on third and fourth instar alatiform aphid larvae (JOHNSON,1959 ; JOHNSON and BIRKS, 1960) although the same authors did not find that severe wounding of adults caused the production of apterous young. Wounding is known to stimulate CA activity in other insects (NEEDHAM, 1965) but the relationships between regeneration, CA activity and prothoracic gland activity are complex (O’FARRELL et al., 1960). It is quite likely that severe wounding of the parent, without decapitation, might not be sufficient to affect the form of the young. Decapitation, however, is much more drastic than any kind of cutting or burning of the thorax or abdomen, and destroys many organ systems (e.g. nervous system, alimentary canal, and dorsal aorta). In particular it destroys the brain and corpus cardiacum/CA system of the parent. Any stimulus to raised CA activity (e.g. from wounding) can thus be received only by the embryos. It is difficult to test this suggestion satisfactorily, because the young of decapitated parents are born prematurely and the young of wounded parents are born after a delay, and they are therefore not equivalent in size or physiological condition. However, it can be seen from this discussion that it is not necessary to postulate an ‘alata-determiner’ to account for the results of decapitation experiments. Acknowledgements-The author thanks Dr. D. T. timON of Sydney University, and Professor K. P. LAMB of the University of Papua and New Guinea, for their advice during the course of this work, and for their helpful criticism of the manuscript. Most of the work described was carried out in the School of Biological Sciences, Sydney University, during the tenure of a C.S.I.R.O. Postgraduate Studentship. REFERENCES DADDR. H. and MITTLER T. E. (1965) Studies on the artificial feeding of the aphid Myers persicue (Sulser)-III. Some major nutritional requirements. J. Insect Physiol. 11, 717-743. HIGHNAMK. C. and HA~KELLP. T. (1964) Th e endocrine systems of isolated and crowded Locusta and Schistocerca in relation to o&zyte growth, and the effects of flying upon maturation. y. Insect Physiol. 10,849-864. JOHNSONB. (1954) Effect of flight on behaviour of Aphisfabae Stop. Nature, Lond. 173,831. JOHNSONB. (1957) Studies on the degeneration of the Ilight muscles of alate aphids-I. A comparative study of the occurrence of muscle breakdown in relation to reproduction in several species. J. Insect Physiol. 1, 248-256. JOHNSONB. (1959) Effect of parasitisation by Aphidius platensis Brethes on the developmental physiology of its host Aphis craccivora Koch. Entomologia exp. appl. 2, 82-99.
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JOHNSON B. (1965) Wing polymorphism in aphids-H. Interaction between aphids. Entomologia exp. appl. 8, 49-64. JOHNSONB. (1966a) Wing polymorphism in aphids-III. The influence of the host plant. Entomologia exp. appl. 9, 213-222. JOHNSONB. (1966b) Wing polymorphism in aphids-IV. The effect of temperature and photoperiod. Entomologia exp. appl. 9, 301-313. JOHNSONB. and BIRKS P. R. (1960) Studies on wing polymorphism in aphids-I. The developmental process involved in the production of the different forms. Entomologia exp. appl. 3, 327-339. KAWADAK. (1965) The development of winged forms in the cabbage aphid, Brevicoryne brassicae Linnaeus-II. The period of determination of wing development. Ber. Ohara Inst. landw. Biol. 13, l-5. KENNEDYJ. S. and STROYANH. L. G. (1959) Biology of aphids. A. Rev. Ent. 4, 139-160. KITZMILLERJ. B. (1950) The time interval between determination and differentiation of wings, ocelli and wing muscles in the aphid Macrosiphum sanborni (Gillette). Am. Nut. 84, 23-50. LAMB K. P. (1956) Physiological relations between aphids and their host plants. Ph.D. Thesis, University of Cambridge. LAMB K. P. and WHITE D. (1966) Effect of temperature, starvation and crowding on production of alate young by the cabbage aphid (Brevicoryne brassicae). Entomologia exp. appl. 9, 179-184. LA~EENCE P. A. (1966) The hormonal control of the development of hairs and bristles in the milkweed bug, Oncopeltus fasciatus Dall. J. exp. Biol. 44, 507-522. LEES A. D. (1961) Clonal polymorphism in aphids. Symp. R. e-nt. Sot. Lond. 1, 68-79. LEES A. D. (1966) The control of polymorphism in aphids. Adw. Insect Physiol. 3,207-277. LEES A. D. (1967) The production of the apterous and alate forms in the aphid Megoura viciae Buckton, with special reference to the r6le of crowding, J. Insect Physiol. 13, 289-318. NEEDHAMA. E. (1965) In Regeneration in Animals and Related Problems (Ed. by KIORTISV. and TEAMPUSCHH. A. L.). North Holland, Amsterdam. O’FAEEELL A. F., STOCKA., RAE C. A., and MORGANJ. A. (1960) Regeneration and development in the cockroach Blatella germanica. C?as. Est. Spol. ent. 57, 317-324. SHULL A. F. (1938) Time of determination and time of differentiation of aphid wings. Am. Nat. 72, 170-179, WHITE D. (1965) Changes in size of the corpus allatum of a polymorphic insect. Nature, Lond. 208, 807. WHITE D. F. (1968) Cabbage aphid: effect of isolation on form and on endocrine activity. Science, Wash. 159, 218-219. WIGGLE~WORTH V. B. (1954) The Physiology of Insect Metamorphosis. Cambridge University Press, Cambridge.