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J. Embryol. exp. Morph. Vol. 22, 1, pp. 27-44, August 1969
Printed in Great Britain
27
The programming of differentiation and its
control by juvenile hormone in saturniids
By JUDITH H. WILLIS 1
From the Department of Entomology, University of Illinois
Although considerable progress has been made with the chemistry of juvenile
hormone (Dahm, Roeller & Trost, 1968), studies on its mechanism of action in
immature insects are still in a preliminary stage. Much of the recent work has
been interpreted as showing an effect of juvenile hormone on the morphogenetic
program through which an insect passes in the course of its ontogeny (Williams,
1961). It is the purpose of this paper to describe three studies which illustrate
the complex nature of this developmental program in saturniid moths.
MATERIALS AND METHODS
Experimental animals
The saturniids {Antheraea polyphemus, Samia cynthia and Hyalophora
cecropia) used in the present study were reared or purchased from dealers in
the United States and England. Staging of animals was carried out by examining
the state of the epidermis and the differentiation of adult structures through the
pupal cuticle as described by Schneiderman & Williams (1954). Partially purified
extracts of juvenile hormone (Williams, 1956) were injected into polyphemus
pupae shortly before the onset of adult development. The amount injected
varied with the preparation and purification of hormone used. Pupae which
receive extra, active corpora allata or juvenile hormone at the onset of adult
development, moult into another pupal instar or a form with some pupal and
some adult characteristics. All pupal-adult intermediates which had a pupa-like
abdomen and a score of + + + or higher in the Williams assay (Williams, 1961)
will hereafter be designated as deuteropupae instead of the more confusing term
'second pupae' which has been used previously.
The pupa-deuteropupa transformation is more rapid than the pupa-adult
transformation (Gilbert & Schneiderman, 1960). Thus, in this study, deuteropupae have been compared with pharate adults as well as with adult moths
because any reduction in size or maturity of an organ in the deuteropupa may
1
Author's address: Department of Entomology, University of Illinois, Urbana, Illinois.
61801, U.S.A.
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J. H. WILLIS
be due to an insufficient growth period and not to a more fundamental difference
in the hormonal environment (see Williams, 1968).
Histological preparations
Whole mounts of cuticle were prepared by removing the adhering scales and
epidermal cells, dehydrating in ethanol, clearing in xylene and mounting on a
slide in Canada balsam or 'HSR'. When the epidermis was to be studied in
whole mounts, it was fixed and stained with Feulgen or hematoxylin prior to
mounting.
Cuticle and compound eyes were fixed in Bouin's or Zenker's fluid; passed
successively through ethyl cellosolve, methyl benzoate, and benzene; embedded
in paraffin wax or 'Paraplast'; and sectioned at 6 /t. Cason's stain (Cason, 1950)
was principally used. The eyes of six polyphemus deuteropupae and several
normal polyphemus and cynthia pupae and developing adults were examined
in serial section.
Autoradiography was performed on sections cut from tissues generously provided by Dr Blair Bowers from animals used in previous work (Bowers &
Williams, 1964) and their methods were followed.
RESULTS
The presence of 'larval' tubercles on untreated pupae
The fifth-instar cecropia larva is elaborately decorated with orange, yellow,
and blue tubercles. A remnant of these larval tubercles can be found in all
cecropia pupae in the form of cells which correspond in position and color to
the tubercles of the larva. These colored cellular patches are especially obvious
in the newly moulted, untanned pupa but small clusters of the blue cells have
also been observed in fully tanned, diapausing pupae.
Occasionally cecropia pupae are found which bear raised knobs of brown
pupal cuticle in the same position in each segment as is occupied by a tubercle
in the larva. This resynthesis of a larval character in a pupa was seen in a most
dramatic form in a batch of cecropia purchased from Mr S. E. Ziemer, a
Wisconsin dealer, in 1959. Fourteen per cent of his 491 pupae possessed tubercles,
and in this respect resembled a larval-pupal intermediate. The abnormal pupae
varied from those which had a few brown tubercles to some which had a full
complement of pigmented tubercles.
These protuberances were an integral part of the heavily sclerotized pupal
cuticle, for after the formation of the moth they were present on the pupal
exuviae. From a study of the cast skins of the final larval instar recovered from
the cocoons of the abnormal pupae, it was evident that these larvae had had
tubercles identical to those found on normal final instar animals.
There was no obvious cause of these abnormalities. No evidence for any
simple genetic basis was provided by the equal distribution of the abnormality
Juvenile hormone in saturniids
29
between the sexes or by a cross between one pair of adults reared from abnormal
pupae. In response to a questionnaire, Mr Ziemer could report no conditions
of diet or environment which might have been responsible for this phenomenon.
Pupae with'larval'tubercles have subsequently been produced experimentally,
either by infecting larvae with Nosema (C. M. Williams, personal communication), a microsporidian which produces juvenile hormone (Finlayson & Walters,
1957; Fisher & Sanborn, 1962), or by injecting larval tubercles with juvenile
hormone or active corpora allata (Staal, 1967). Thus, although we have no
direct evidence, it seems reasonable to suspect that the abnormal Ziemer pupae
were caused by Nosema infection, and thus indirectly by an excess of juvenile
hormone.
Many of the adults which emerged from the tubercle-bearing pupae had one
significant abnormality: minute scaleless patches on the dorsal side of the
abdomen in the same location as the tubercles of the pupae. No adult from a
normal pupa has ever been observed to have such scaleless areas. A microscopic
examination of whole mounts of the adult cuticle of these moths revealed that
some of these patches consisted of scaleless thickened, tanned 'pupal' cuticle
surrounded by a thin adult scale-bearing cuticle (Fig. 4D). In one case a heavily
tanned tubercle-like protuberance was found on the adult cuticle of an animal
which had had a complete set of tubercles in the pupal stage.
Development of the compound eye under
the influence of juvenile hormone
Detailed descriptions of the histology and developmental significance of the
various parts of the lepidopteran compound eye are available in the literature
(Urnbach, 1934; Wolsky, 1949). I have examined the histology of the eyes of
normal diapausing and adult cynthia and polyphemus, and pharate adults of
cynthia on days 3, 7, 8, 11, 17 and polyphemus on day 13 after the initiation of
adult development.
The normal pupal eye
In Lepidoptera, bilateral crescents of smooth cuticle exhibiting rudimentary
facets are located on the head of the pupa and are partially or completely covered
by the antennae. These crescents face outward and are limited on their anteromedial borders by a dark unfaceted furrow (Fig. 3 A). In the diapausing pupa,
the cells which underlie the crescent near the furrow are already organized into
clusters of undifferentiated cells, each cluster destined to form an individual
ommatidium (Umbach, 1934) (Fig. 1A). The rest of the presumptive eye is
present as a multilayered epithelium which lies anterior to the crescent and which
is not organized into cell clusters (Wolsky, 1949).
Eye development in the pharate adult
The cells underlying the crescent are not only morphologically more advanced
in their differentiation than the rest of the presumptive eye, but, as Wolsky
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J. H. WILLIS
(1949) has shown by extirpation and microcautery, this region also serves as a
'differentiation center' which is required for the peripheral regions to differentiate. Umbach (1934) reports that approximately half-way through adult
development the peripheral regions of the developing eye 'catch-up' with the
differentiation center and from then on all of the ommatidia differentiate synchronously. I have found that the same pattern of development is present in
cynthia where synchronous differentiation is established by the seventh day of
adult development, at a time when the cornea has not yet appeared (Fig. 1E).
By the thirteenth day of adult development the eyes of polyphemus have
assumed their final form, although considerable enlargement by virtue of
increase in the size of the individual cellular elements still occurs during the
remaining week before emergence (Fig. 1F).
The adult eye
Although the development and morphology of the adult saturniid eye are
similar to that described by Umbach (1934) for Ephestia, a brief illustrated
(Fig. 1A-F) account of my findings is included in order to facilitate a consideration of the abnormalities found in the deuteropupa. Umbach's terminology is
used throughout.
The adult eye is covered with a transparent cornea composed of hexagonal
facets each of which covers an individual ommatidium (Fig. 3C). Directly
beneath the cornea lie Semper's cells, which secrete the cuticular crystalline
cone. The cornea is formed by the corneal pigment cells whose nuclei lie lateral
and proximal to Semper's cell nuclei. The retinula cell nuclei lie in a cluster
connected distally to the crystalline cone by an axial strand and proximally to the
rhabdom by another thin filament (Achsenfaden). The rhabdom is surrounded
ABBREVIATIONS
bm, Basement membrane; c, cornea; cc, crystalline cone; r, retinula cell nuclei;
S, Semper's cell nucleus; /, tracheoles of tapetal layer. The line in each figure
represents 20/£.
Fig. 1. Normal eye development
A. Diapausing polyphemus pupa. Cross-section of the eye showing the cell clusters
(arrows) which give rise to individual ommatidia.
B. Cynthia: third-day adult development. The crystalline cones are forming.
C. Autoradiograph of a cynthia eye on day 2 of adult development. The arrow
indicates the silver grains at the border of the eye. The region to the left of the
silver grains had begun differentiating.
D. Polyphemus adult. Cross-section through rhabdoms with surrounding tracheoles
of tapetal layer.
E. Cynthia: seventh day of adult development. The crystalline cone vacuoles have
fused. The arrow designates the groove which delineates the edge of the eye.
F. Polyphemus: thirteenth day of adult development. The eye has the adult configuration but has not yet reached full size.
Juvenile hormone in saturniids
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J. H. WILLIS
Juvenile hormone in satumiids
33
distally by other (Neben) pigment cells and proximally by the tapetal layer,
a group of tightly packed tracheoles (Fig. 1 D). At the base of each ommatidium
lies the basal retinula cell. The eye is covered at its base with a basement membrane and laterally is separated from the rest of the head by the cuticular eye
capsule.
The eye of the deuteropupa
(1) Gross structure
It is an interesting curiosity, first reported by Williams (1959), that no matter
how much juvenile hormone is injected into a pupa and no matter how completely pupal most of the characteristics of the resulting deuteropupa are, their
eyes never fail to assume some of the characteristics of adult eyes. The most
obvious difference between the compound eye of the adult and the eye of the
deuteropupa is the smaller size of the latter. The size of the compound eye is
correlated with the degree of formation of pupal structure (Gilbert & Schneiderman, 1960; Williams, 1961), so that, in cases where the deuteropupa is extremely
1
pupal', the compound eye may appear no larger than the initial pupal eye
crescent. The severity of internal abnormalities was also positively correlated
with the retention of general pupal characteristics.
(2) General organization and number of elements
By the seventh day of adult development in cynthia, presumptive eye tissue
is set off from the adjacent epidermis by a deep fold of cells which subsequently
is covered with cuticle which forms the eye capsule (Fig. 1 E). In the deuteropupa,
the sharp distinction between eye and non-eye is lacking, the eye region of the
deuteropupa consisting of a central area composed of recognizable pigmented
ommatidia, a lateral zone of clustered poorly differentiated cells organized into
presumptive ommatidia (Fig. 2B) and a peripheral zone of a multilayered
epithelium. A shallow furrow can be recognized peripherally to these regions
(Fig. 2A). This pattern suggests that those cells which were organized into
ommatidial groups in the pupa (the cells underlying the pupal eye crescent)
cannot be completely blocked in their course of differentiation by juvenile
hormone. By contrast, the peripheral regions (which were not organized into
pre-ommatidial groups in the pupa) either remain as they were or, in the case of
deuteropupae with large eyes, differentiate with some cells only reaching the
stage normally reached by the central region immediately following pupation.
Fig. 2. Deuteropupa eye
A. Note the extensive growth zone (between arrows) at the edge of the eye.
B. The growth zone from another deuteropupa. The arrow indicates a cell cluster.
A maximally formed crystalline cone is present immediately adjacent to the growth
zone.
C. Central region from a more adult-like deuteropupa. The major elements of the
eye are recognizable.
3
J E E M 22
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J. H. WILLIS
Thus, in the formation of a second pupal instar, the central and peripheral
regions each take but one step forward in development.
The pupal eye consists of approximately 2000 facets, the adult eye of about
8000 ommatidia. The smallest deuteropupal cornea examined (Fig. 3B) had
roughly 4000 modified hexagonal corneal lenses; the region which appeared
most like the crescent of the normal pupa contributed an additional 2000 rudimentary facets. The correspondence in number of rudimentary facets in the
crescent of the deuteropupa and the normal pupa may be due either to a
mechanism which governs crescent size or to the recruitment of new cells to
form the ommatidia of the deuteropupa leaving the original crescent cells
unaltered. The latter interpretation is highly unlikely in view of the experimental
work which has shown that the 'differentiation center' must undergo some
differentiation before the other areas of the eye (Wolsky, 1949).
(3) Histology of the ommatidium
(a) Length of the ommatidia. There was considerable variation in the length
of the ommatidia in the deuteropupae, and the length of the ommatidia was
positively correlated with the size of the eye. One deuteropupa's ommatidia
Table 1. The size of components of the eye
in polyphemus—maximum dimension
Pharate adult
Structure
Crystalline cone (length, /*)
Ommatidia (length) exclusive
of cornea (/*)
Deuteropupa
Adult
Day 13
Most
pupal-like
Least
pupal-like
65
549
54
254
20
137
29
245
were but one-quarter the length found in an adult, and the least affected
deuteropupa had ommatidia which were the same length as are normally
encountered two-thirds of the way through adult development (Table 1;
Fig. 2 A, C).
Fig. 3. Whole mounts of cuticle
A. The pupal eye. The unfaceted furrow lies to the left and the area of presumptive
ommatidia to the right.
B. The deuteropupal eye. The equivalent of the pupal eye crescent is in the top of the
picture, the region with abnormal facets lies below.
C. The cornea of an adult polyphemus.
D. The cornea of a deuteropupa. Note the abnormality in the center of each
irregular facet.
E. Polygonal field zone of a pupa.
F. Polygonal field zone of a deuteropupa at the same magnification as E.
35
Juvenile hormone in saturniids
mm
3-2
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J. H. WILLIS
(b) The cornea. The most adult-like deuteropupal corneal facets were rounded
hexagons and contained a central protuberance; their diameter was smaller
than the diameter of the hexagons of the normal adult eye (Fig. 3D). In many
of the deuteropupae a distinct pupal eye crescent was present in addition to the
abnormal facets (Fig. 3B).
(c) The crystalline cone. Frequently the only indications of the crystalline
cone in the deuteropupa were unfused vacuoles of secretion in Semper's cells
(Fig. 2 A). Even when the crystalline cone was present as a fused single structure,
it was only about half the length normally reached by the thirteenth day of adult
development (Table 1). The irregularities and small size of the cones cannot be
due to premature emergence alone, for by the seventh day of adult development
in cynthia complete fusion of the vacuoles into a single element has occurred
(Fig. IE).
(d) The retinular cells and rhabdom. Frequently, there was no indication of
the rhabdom in the deuteropupa, and when it was present it generally extended
only part way to the basement membrane (Fig. 2 A, C). The mature form of the
rhabdom (Fig. ID) was not evident in cross-sections until the fourteenth day
of adult development. In the deuteropupa the rhabdom was never represented
by more than a dense rod in cross-section.
The clustered retinula cell nuclei are apparent in the eye of the deuteropupa
but the basal retinula cell could not be located in many of the preparations
(Fig. 2C). Presumably, it had not yet migrated away from the distal nuclear
cluster, although this migration is normally complete by the eleventh day of
adult development in cynthia (Fig. IE). Furthermore, in the deuteropupa the
mass of retinula cell nuclei was located just below the crystalline cone. In the
few cases where they were at a slight distance from the cone, the axial strand
was recognizable.
(e) The tapetum and outer pigment cells. There was no indication of a tapetal
layer in the eyes of the deuteropupa. The area between the crystalline cone and
basement membrane was filled only by pigment cells (Fig. 2C). These cells,
which appear as a matrix of triangles with pigmented sides when viewed in
cross-section, were normally restricted to the area between the crystalline cone
and the distal end of the retinula cell nuclear cluster (Fig. 1F). The tapetal layer
was not evident in normal cynthia until the fourteenth day of adult development.
(/) Nervous elements. Nerves extend to the basement membrane across the
entire width of the deuteropupal eye including the peripheral area, which had
only differentiated as far as the cell-cluster stage (Fig. 2 A).
(4) Incorporation of tritiated thymidine into developing eyes
An examination was made of autoradiographs of normal eyes from a cynthia
on the second day of adult development which had been injected with tritiated
thymidine and fixed 18 h later. Although there was general background labelling
over the entire eye, none of the eye elements which showed obvious signs of
Juvenile hormone in saturniids
37
differentiation had any conspicuous labelling over their nuclei. By contrast,
there was heavy labelling at the peripheral borders of the eye in the regions where
the stratified epithelium had not yet formed cell clusters (Fig. 1C). Thus, while
the peripheral borders of the eye incorporate thymidine and presumably synthesize DNA prior to forming ommatidia, the previously organized central
region, which is insensitive to juvenile hormone, has already completed DNA
synthesis.
Abdominal cuticle
A more direct analysis of the progressive differentiation of individual cells
has been possible by utilizing the epidermis of the abdomen. The virtues of the
insect epidermis for developmental analysis are its uniformity in cell type, singlelayered nature, and well-documented developmental activities (Wigglesworth,
1959). Especially well suited for analysis is the anterior region of the intersegmental membranes of lepidoptera, where each epidermal cell secretes a discrete
'polygonal field' which is, in fact, a 'cast' of the surface of the underlying cell
(Kiihn & Piepho, 1940) (Figs. 3E, F, 4A-C). The area and density of the polygonal fields therefore reveal the surface area and density of the underlying
epidermal cells.
When a deuteropupa is formed following the injection of juvenile hormone,
it remains within the exuviae of the old pupal cuticle. One may punch a small
area of cuticle from the deuteropupa along with the overlying bit of cuticle
from the first (normal) pupa. Marcus (1962) reports that in Galleria some of the
cells of this region of the intersegmental membrane die, but that there is no cell
division between the pupal and adult stages. Consequently, it seems likely that
in the present work the two cuticles are secreted by the same group of cells.
Analysis of these double discs of cuticle revealed that there were approximately half as many cells per unit area in the deuteropupal cuticle as there were
in the original (Fig. 3 E, F). The surface of the polygonal fields of the deuteropupa
looks more irregular in texture than that of the pupa.
A comparison of cross-sections of cuticle with its epidermis in diapausing
pupae (Fig. 4A, B) and deuteropupae (Fig. 4C) also revealed an increased cell
size in the latter. The height of the epidermal cell layer in the diapausing pupa
was only about 13 jti, while in the deuteropupa the cell layer may reach 47 /i in
height. The cuticle was thicker in the diapausing pupa (up to 78 fi) than in the
deuteropupa (maximum 50 /a). The nuclei of the epidermal cells were also
significantly larger in the deuteropupa than in the diapausing pupa (Fig. 4B, C).
The intense Feulgen staining, the presence of extra nucleoli, and the greater
nuclear size all suggest that these cells were polyploid in the deuteropupa.
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J. H. WILLIS
Juvenile hormone in saturniids
39
DISCUSSION
Action of juvenile hormone
(1) Theories
Ever since the discovery by Wigglesworth (1936) of the profound morphogenetic control exerted by juvenile hormone there has been keen interest in its
mode of action. Current hypotheses as to the primary site of its activity favor
the nucleus. It has been postulated that juvenile hormone selects a set of genes
which then direct the appropriate syntheses. Present conjecture is centered on
whether the hormone activates a set of 'larval genes' (Wigglesworth, 1959) or
blocks derepression of 'new' genes thereby enforcing rereading of a set of
'previously used genes' (Williams, 1961). Since it has been shown (Nayar, 1954)
that larval cuticle grafted on to pupae will form scales and not pupal cuticle
when the pupa moults to an adult, neither hypothesis can require that all steps
in the program be morphologically expressed.
The distinction between these superficially similar hypotheses is of fundamental significance. We can diagram the life-history of an insect with three
larval instars, a pupal, and an adult stage as follows:
x a b c/x a b cjx abc dejxfg h ij/x k I in no
larvax larva2 larva3
pupa adult
A- represents the numerous syntheses which are common to all stages. The
results following an injection of excess juvenile hormone into a pupa (between
stagesy and k) can be predicted from the two hypotheses, thus making it possible
to distinguish between stringent interpretations of the hypotheses. If the
Wigglesworth hypothesis is operative, larval syntheses (xabc) should follow
such treatment. According to the Williams ('status quo') hypothesis the pupal
syntheses (xfghij) would be repeated. Unfortunately, actual systems have not
responded to hormonal manipulation in such an unambiguous manner. Furthermore, Wigglesworth (1964, 1965) feels that developmental 'inertia' and evolutionary pressures may modulate the response to juvenile hormone.
Reversal of metamorphosis, i.e. reversion to the larval condition at the pupal
adult moult, has never been demonstrated in any saturniid. However, there are
rare but well-documented cases of just such reversals in a few other systems
(Wigglesworth, 1940; Piepho & Meyer, 1951; Lawrence, 1966). The 'status quo'
FIGURE4
A. The polygonal field zone of a pupa in cross section.
B. The epidermis from the same region.
C. The polygonal field zone and adhering epidermis from a deuteropupa.
D. Whole mount of cuticle from a moth which had tubercles as a pupa. Note the
central structure which resembled tanned pupal cuticle and the surrounding
socketless region.
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J. H. WILLIS
hypothesis requires that the developmental program contains some mechanism
whereby the cells keep track of their progress through the program. These cases
of reversal of metamorphosis are not, however, a deciding factor in distinguishing between the Wigglesworth and Williams hypotheses. It is possible to account
for the reversals reported in the literature by postulating that the cells have 'lost
track' of their temporal progress through the developmental program in much
the same manner that imaginal disc cells of Drosophila have been 'transdetermined', i.e. lost their state of spatial determination, by prolonged culture in
adult hosts (Hadorn, 1966). It is significant that the same conditions which are
necessary for transdetermination, namely prolonged culture with considerable
cell multiplication, are identical to the conditions which have produced reversal
of metamorphosis.
(2) Evidence for a program
The results presented in this paper constitute evidence for a developmental
program intrinsic to the individual cells. Thus the larval tubercles which were
found on pupae remained out of phase with the rest of the organism; in short,
the cells in question secreted larval cuticle on a pupal background, and pupal
cuticle on an adult background. The study of the eye demonstrated that the cells
of the pupal eye field are relatively insensitive to juvenile hormone while the
anterior region, which was not yet organized into pre-ommatidia in the pupa,
was held in check and could not complete all of adult differentiation. Indeed the
eyes of a deuteropupa resemble those of a hemimetabolous insect such as
Notonecta (Liidtke, 1940), which has a growth zone around the periphery of the
nymphal eye which contributes to the new facets added at each instar. The
recognition of the stepwise differentiation of the eye explains some of contradictory results in the literature. Gilbert & Schneiderman (1960) attribute to species
differences their finding that the pupal eye of polyphemus is insensitive to
juvenile hormone whereas Piepho (1942) had shown that the eye discs of Galleria
were one of the most sensitive tissues when corpora allata were implanted into
last instar larvae. An alternative explanation is that certain regions of the pupal
eye have reached a stage in their program which is relatively insensitive to the
juvenile hormone whereas the imaginal disc of the larval eye is still at a state in
its program which is highly sensitive. This interpretation is strengthened by the
findings of Tsao, Jou & Chiang (1963), who showed that injection of juvenile
hormone extracts into non-diapausing Samia ( = Attacus) ricini pupae within
12 h after pupation resulted in eyeless deuteropupae. In addition to the obvious
completion of the cuticle, Bowers & Williams (1964) have demonstrated that
there is DNA synthesis in the epidermal cells of the head during this period.
These findings suggest that there must be some progress in the developmental
program of the eye prior to the onset of diapause.
Juvenile hormone in saturniids
41
(3) What is the sensitive step in the program?
It is thus evident that individual tissues can proceed in the developmental
program independently of adjacent tissues, and with differential sensitivities to
juvenile hormone at different stages in their development. Furthermore, the
program is responsible for a dissimilar array of syntheses in different tissues. In
the general body epidermis, the presence of excess juvenile hormone favors the
resynthesis of highly pigmented thin larval cuticle at the larval-pupal moult and
heavily tanned pupal cuticle at the pupal-adult moult. Sensitive regions of the
eye are prevented from carrying out diverse synthetic acts which are performed
in the identical hormonal environment by cells which are more advanced but
still morphologically undifferentiated.
Although the completion of DNA synthesis appears to be one factor which
makes the central region of the eye relatively insensitive to juvenile hormone,
both DNA synthesis and mitosis can be ruled out as sites of that hormone's
action. The present study shows that post-mitotic tissues such as the intersegmental membrane are sensitive to juvenile hormone; and Wigglesworth
(1959) demonstrated that juvenile hormone increases mitotic activity in specific
regions of Rhodnius. The polygonal field zone apparently becomes polyploid
under the influence of juvenile hormone; and Krishnakumaran, Berry, Oberlander & Schneiderman (1967) could detect no differences between tritiated
thymidine incorporation during the pupal-adult and pupal-deuteropupal moults.
(4) Can the action of juvenile hormone be delayed?
Recent studies with embryos (Riddiford & Williams, 1967) and with developing aphids (White, 1968) have shown that juvenile hormone applied at one stage
may not show an effect until several instars later. Riddiford and Williams applied
juvenile hormone preparations to developing saturniid embryos and found that
larval development was normal but that the pupae frequently had 'larval'
tubercles and were similar to those described in the first section of this paper.
They interpreted their data to mean that the epidermal cell program had been
'reset'. There is an alternative explanation to this delayed action of the responsive cells, namely that extra juvenile hormone in the embryo might prevent the
slowing down of the activity of the corpora allata which normally precedes
pupation. In that case abnormal pupae would be produced because the moult
took place in the presence of excess juvenile hormone. The permanent alteration
of endocrine activity by prenatal treatment with hormones is well documented
in vertebrates (Barraclough, 1967).
The significance of the abnormalities in the deuteropupal eye
Although the deuteropupal eye bears a superficial resemblance to the adult
eye it does not show normal morphology. Thus the tapetal layer is not formed
and the rhabdom is not completely differentiated. The failure of the ommatidia
42
J. H. WILLIS
to differentiate completely may be due in part to the precocious maturation of
the deuteropupa—especially to the precocious secretion of new cuticle. It is
unlikely that the abnormalities are caused by any failure of the central nervous
system, for Wolsky (1949) has shown that the eyes of Bombyx adults deprived
of brains as young pupae are usually completely normal. Furthermore, corneae
from cecropia adults debrained as pre-diapausing pupae are also normal (unpublished observations). The abnormalities of the deuteropupal eye are essentially different from the temporally graded abnormalities produced by the
implantation of 5-fluorouracil in Ephestia (Imberski, 1967). Possible insight
into the nature of the abnormalities might be obtained by comparing the eye of
true deuteropupae with those from the abnormal adults obtained by Williams
(1968) following injections of excessive amounts of ecdysone and some of its
analogues into pupae. He has attributed the resulting abnormalities to a speed-up
in developmental rate and premature cuticle deposition.
The finding that certain elements of the pupal eye, such as the protuberance
in the center of each corneal facet, are maintained while other elements differentiate towards the adult structure, is reminiscent of the findings of Wigglesworth (1940) that juvenile hormone applied at successively later times in the
nymphal-adult transformation of Rhodnius affected a decreasing number of
characters. These findings show that juvenile hormone does not exert an all-ornone action, but that it can control a developmental sequence within an individual cell. Juvenile hormone's precise control over such small steps in the
developmental program constitutes strong evidence for the status quo hypothesis.
SUMMARY
1. Cecropia pupae with larval tubercles and their transformation into adults
with patches of scaleless or pupal cuticle were described.
2. Eyes of deuteropupae produced by the injection of juvenile hormone into
polyphemus pupae were analysed. The deuteropupal eye was smaller than the
normal adult eye, was bordered by a 'growth zone', and individual elements of
the ommatidia had not differentiated normally or completely.
3. The polygonal field zone of the intersegmental membrane of the deuteropupa consisted of fewer and larger cells than the corresponding region of the
pupa.
4. These results were interpreted as supporting the 'status quo' theory of
juvenile hormone's action.
Juvenile hormone in saturniids
43
ZUSSAMENFASSUNG
Der Ablauf der Differentiation und seine Kontrolle durch
das Juvenilhormon bei Saturniiden
1. Cecropia-Puppen mit larvalen Tuberkeln und deren Umformung in adulte
Tiere mit Flecken von schuppenloser oder pupaler Kutikula wurden beschrieben.
2. Untersucht wurden die Augen der Deutero-Puppen, die durch die Injektion
des Juvenilhormons in Polyphemus-Puppen entstanden. Das deutero-pupale
Auge war kleiner als das normale Auge eines adulten Tieres, es war begrenzt
durch eine 'Wachstums-Zone', und einzelne Teile der Ommatidien hatten sich
nicht in der normalen Weise oder vollstandig ausdifferenziert.
3. Die polygonale Feld-Zone in der intersegmentalen Membran der DeuteroPuppen bestand aus weniger und grosseren Zellen als die entsptrechende Zone
einer Puppe.
4. Diese Ergebnisse wurden so interpretiert, dass sie die 'Status gwo'-Theorie
der Wirkungsweise des Juvenilhormons unterstiizen.
This work has been supported by grants CF-8605 and HD-03163 of the U.S. Public Health
Service. Part of the work was carried out in the Department of Zoology, Oxford University,
and the hospitality of Dr Peter Brunet is gratefully acknowledged. Dr Ellis MacLeod provided useful criticism of an early draft of the manuscript. My special thanks are due to
Professor Carroll M. Williams. This study was begun under his direction at Harvard University, and he has continued to provide stimulating suggestions throughout the course of the
work and the preparation of the manuscript.
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