J. Embryol. exp. Morpli. Vol. 21, I, pp. 1-21, February 1969
Printed in Great Britain
Morphogenetic analysis
of the effects of juvenile hormone analogues and
other morphogenetically active substances on
embryos of Schistocerca gregaria (Forskal)
By VLADIMIR J. A. NOVAK1
From the Department of Physiology, Institute of Entomology,
Czechoslovak Academy of Sciences, Prague
One of the basic assumptions of the author's gradient-factor theory of insect
morphogenesis is that the effects of the hypothetical gradient-factor on tissue
growth can be reproduced by the juvenile hormone, not only during postembryonic development, but also in the course of embryogenesis (Novak,
1951 a, b, 1956, 1966). This concept was originally based on the more or less
indirect evidence supplied by the findings of Pflugfelder (1947) in Dixippus
morosus and by those of Novak (1951 b) in Oncopeltus fasciatus.
Recently, however, direct evidence has been made available by the work of
Slama & Williams (1966) on Pyrrhocoris apterus and of Riddiford (1966) on
Hyalophora cecropia. Using the 'paper factor' in the first case and cecropia oil
in the second, the above authors succeeded in demonstrating that these substances were able to block embryogenesis when applied to the egg surface or
to the female before egg-laying commenced. Most of the pathological morphogenetic effects associated with inability to hatch were obtained with only minute
amounts of the given substances. The results indicate that these substances
ought to prove very effective as ovicides in actual insect pest control. No attempt
has been made to explain the mechanism of their action on embryos, however,
or to compare it with their effect on larvae.
Locusts provide suitable material for the study of insect embryogenesis and
of factors affecting it, both because of their large size and of their high resistance
to many types of experimental treatment. By exposing embryos of Schistocerca
gregaria to the action of the corpus allatum hormone and by applying farnesol
methyl ether and other JH analogues to the egg surface at different intervals
during morphogenesis, the author succeeded in obtaining a wide variety of
embryonic morphogenetic aberrations of varying kinds and degrees (Novak,
1969a).
1
Author's address: Odd. Fysiologie, Entomologicky Ustav CSAV, Na Folimance 5,
Praha 2, Czechoslovakia.
I
JEEM2I
V. J. A. NOVAK
The aim of the present paper is to classify these morphogenetic effects. It also
attempts to explain the findings and the mechanism of the action of the substances employed in terms of the author's gradient-factor theory.
MATERIAL AND METHODS
Eggs of Schistocerca gregaria laid in glass tubes 12 cm long and 4 cm in
diameter, filled with moist sterilized sand, were incubated at 32 °C; at this
temperature they hatch in 12 days. Experimental eggs were washed in water
containing 1 % nipagin to prevent the growth of moulds and small amounts of
penicillin and streptomycin to prevent bacterial infections; they were then placed
in Petri dishes on several sheets of filter paper moistened with the same
solution.
The chemicals to be tested were applied to the egg surface as drops by means
of a thin capillary tube. As most of them were lipophilic in character, the egg
surface had to be roughly dried just before applying them. Solid substances
were either dissolved in an appropriate solvent, using the solvent only as the
control, or a small particle was applied to the egg surface with the tip of a needle,
followed by a drop of the solvent from the capillary tube.
The following substances were tested:
Farnesol trans-trans (a British product received from Dr D. B. Carlisle).
Farnesol methyl ether (synthesized from the above by Dr D. B. Carlisle in
the laboratories of the Anti-Locust Research Centre, London).
Dihydrochloromethyl farnesoate (synthesized by Dr J. Romariuk of the Institute
of Organic Chemistry, Czechoslovak Academy of Sciences, Prague).
Queen honey-bee extract (ethanol extract of 200 fertilized queens aged 3-5
years, evaporated in vacuo and dissolved in 96% ethanol).
9-oxotrans-2-decenoic acid (synthesized by Dr K. Streibl, Lipids Department,
Institute of Organic Chemistry, Czechoslovak Academy of Sciences, Prague).
9-hydroxy-2-decenoic acid (same source).
lO-hydroxy-2-decenoic acid (same source).
Cecropia oil (extract of male abdomina of Hyalophora cecropia, received from
Professor H. A. Schneiderman, Developmental Biology Center, Western Reserve
University, Cleveland, Ohio, and from Dr G. B. Staal, Institute of Entomology,
Agricultural University, Wageningen, Netherlands).
Paper factor (methanol extract of pages of a journal of American origin
(Scientific American); this was diluted with water and extracted with petroleum
ether, which was afterwards evaporated off (Slama & Williams, 1965).
Control substances: pure 96% ethanol and olive oil.
To study the effects of morphogenetically active substances the arrested eggs
were dechorionated by immersing them in a 2% solution of sodium hypochlorite, usually 1 or 2 days after the control eggs hatched. When thoroughly
rinsed in water, normal eggs continued to develop and hatched without detectable
Juvenile hormone effects
3
injury after this treatment. Only specimens used for histological sections were
dissected from the egg membranes.
Eggs used for paraffin sections were fixed in Bouin's fluid, dehydrated through
the usual series of alcohols, embedded in paraffin after Peterfi's methylbenzoate
treatment and stained after rehydration by Ehrlich's haematoxylin-eosin method.
Selected specimens, after dissection from the egg membranes and fixation,
were preserved in 70% ethanol containing 3% glycerin.
RESULTS
Specimens treated with JH analogues may stop developing at any stage of
embryogenesis, from cleavage of the original egg cell (zygote) to the fully
developed embryo. They can easily be distinguished from normal specimens of
the corresponding stage by the following features:
(i) They are still alive when the controls of the same age have hatched and
often for a long time afterwards.
(ii) They are usually more heavily pigmented and their compound eyes
undergo partial differentiation and become pigmented at a much earlier stage
than in normal specimens.
(iii) Less of the yolk is consumed and the yolk often has a specifically granulated structure.
(iv) When they have reached a more advanced stage of development, the
embryonic moulting process takes place at an earlier stage than in normal
specimens, so that we often find experimental embryos invested with a transparent exuvia which never, of course, hatch.
When estimating the morphogenetic effect of a test substance in a given case,
the following features were taken into account: (a) the occurrence and timing
of the embryonic moulting process; (b) the degree of pigmentation in normal
and abnormal areas; (c) the amount of unconsumed yolk and the size of the
embryo; (d) unsuccessful blastokinesis; (e) absence of the white cuticle; (/) transparency of various areas of the body; (g) the presence of white urate deposits
and their position and amount; (h) the time at which changes occurred in the
experimental specimens (i.e. after application of a test substance), compared
with the time of changes in the controls.
CLASSIFICATION OF MORPHOLOGICAL EFFECTS
The morphological changes produced by the application of active substances
to over 1500 eggs (not counting the controls) form a more or less complete series
of transitions ranging from the arrest of development in the initial stage of
embryogenesis (during cleavage) to the complete development of apparently
normal embryos. It was nevertheless thought that it would be useful, in order
to facilitate the description and analysis of these effects, to arrange them in a
scale of representative types. Changes of a purely teratological character (e.g.
4
V. J. A. NOVAK
asymmetrical development or the obliteration of various organs) were considered
separately. The following classification was therefore used, although, of course,
it must be borne in mind that the individual grades are only arbitrary steps in a
whole series of effects.
A. Symmetrical changes
I. Fully developed embryos without detectable morphological aberrations,
but failing to hatch (Plate 1F).
PLATE 1
Eggs of Schistocerca gregaria treated with farnesol methyl ether and dechorionated with
sodium hypochlorite (2%). A, Normal egg, outset of embryogenesis; B, type XI. (before
blastokinesis); C, abnormal blastokinesis (note granulated yolk); D, type VI (mid-blastokinesis); E, type IV (miniature embryo); F, type I, fully developed embryo.
Juvenile hormone effects
5
II. Embryos with abbreviated appendages and usually with increased pigmentation but no qualitative morphological abnormalities, with complete
dorsal closure and with no yolk (Plate 3H).
III. More or less fully differentiated embryos, but distinctly shorter than the
length of the egg. Communication of the digestive tube with the rest of the yolk
sac through a dorsal opening in the occipital part of the head. Abbreviated
appendages, increased red-brown pigmentation (Plates 2E, 3F).
PLATE 2
Embryos of Schistocerca gregaria dissected from eggs treated with farnesol methyl ether.
A, Type X, cyclopoid embryo with pigmented compound eyes; B, type VII, outset of blastokinesis; C, type VI; D, type V; E, type V, dorsal view (note pigmentation of margins of yolksac opening); F, type IIT, miniature embryo with remains of yolk sac.
V. J. A. NOVAK
PLATE 3
Embryos of Schistocerca gregaria juvenilized with farnesol m. e., dissected from egg. A, Type
X, lateral view (note residue of serosal sac; B, type IX; C, type VII; D, type VI; E, type V;
F, type IV (note marked sclerotization); G, advanced stage of type III (note sclerotic
pleuropods); H, type II.
Juvenile hormone effects
7
TV. Dwarf embryos with incomplete differentiation of segments of antennae
and maxillary palps, tarsi, etc., heavily pigmented. Yolk sac occupying more
than one-third of length of egg and communicating with body via head and
part of thorax (Plates 1 E, 2F).
V. Incompletely differentiated embryos with reduced pigmentation compared
with IV, but greater than in the untreated controls. Incomplete blastokinesis
(end of abdomen bent dorsally) (Plate 3E).
VI. Embryos with more severe morphological defects, often with a reduced
number of abdominal segments, in mid-blastokinesis. Only the compound eyes
partly pigmented (Plates I D , 3D).
VII. Polypod embryos, incipient blastokinesis, head in direction of hydropyle,
towards ventral side of egg. Two-thirds or more of yolk still remaining (Plates
2C, 3C).
VIII. Protopod embryos with undeveloped abdominal appendages, before
blastokinesis. Body in dorsal position, head directed towards hydropyle, only
the compound eyes pigmented (Plate 1B).
IX. Embryos immersed in yolk, only slight differentiation of thorax, abdomen
in form of thin plate. Length of embryo less than one-fifth of that of the egg
(Plate 3 B).
X. Cyclopoid embryos with undifferentiated head and body and no appendages, not more than one-tenth of length of egg. Often connected with yolk by
means of thin tube (formed of serosa?). Compound eyes often distinguishable
and slightly pigmented (Plates 2 A, 3 A).
XI. Blastoderm stage, with more or less distinct germinal band. Round
hydropyle, blastoderm usually detached from white cuticle (Plate IB;
Text-fig. 6).
XII. Pre-blastoderm stage without secondary egg membranes (yellow and
white cuticle). Yolk granulated and usually partly consumed by hypertrophied
cleavage cells (Cf. Plate 1 B, C).
B. Asymmetrical changes
I. Reduction of segments in appendages only.
II. Reduction of parts of body laterally.
III. Reduction of parts of body along anterio-posterior axis.
The types described above are merely arbitrary steps in the continuous series
of morphological changes caused by the action of JH analogues. In addition,
morphologically identical specimens may display a number of profound physiological differences, to be enumerated below. For example, types IV or V may
or may not be in an advanced stage of the moulting process (detached or not
detached from the transparent embryonic cuticle), or may be more or less
pigmented than the controls. Similarly, types VI-IX may differ as regards the
state of blastokinesis, the degree of transparency of the body or the amount of
urates deposited, types X-XII may or may not have a granulated yolk as a
8
V. J. A. NOVAK
result of survival or non-survival of cleavage cells, and may also have urate
deposits, while type X may or may not have distinctly pigmented compound
eyes. Greater or lesser asymmetry may likewise occur in most types. It will
therefore be useful to give a detailed description of several specific individuals
and of the sequence of changes in various parts of the body.
DESCRIPTION
Closing of dorsal body walls and communication with yolksac. Treated specimens
of types III and V may differ as regards the size of the dorsal orifice, which
communicates with the yolk. In some specimens it is limited to the anterior
part of the occiput, while in others it may extend further back, to the thorax
B
Text-fig. 1. Juvenilized embryos with remains of yolk sac communication with
digestive tube through dorsal opening. A, Opening limited to anterior part of occiput
(type III); B, opening in head and pronotum (type IV). Margins of opening
markedly sclerotic. Farnesol methyl ether.
(Text-fig. 1 A, B). Although the size of the orifice varies considerably, it is easy
to see that it is relatively larger, on average, in less differentiated embryos and
that the anterior margin is always on the occiput, while the position of the
posterior margin changes with the size of the opening. This underlines the fact
that in normal development the dorsal walls of the body close from the posterior
to the anterior end of the body.
Growth of appendages. When comparing the state of development of the
antennae, palps, mandibles and legs in individuals, a number of simple rules
Juvenile hormone effects
9
can be demonstrated. The legs are differentiated at quite an early stage of
development and their further fate depends merely on growth in length and
width. The action of the test substances reduces growth in length in favour of
growth in width (Text-fig. 2 A, B). In the mandibles, the teeth are differentiated
Text-fig. 2. Juvenilized embryo with shortened, slightly deformed appendages. A,
Widened and shortened metathoracic leg; B, mandibles: B]5 normal, fully developed
embryo; B2, slightly affected embryo; B3, more severely affected embryo (differentiation stopped at the two teeth stage, but growth continued). Farnesol methyl ether.
Text-fig. 3. Embryos with reduced number of abdominal segments. A, B, With two
abdominal segments; A, in egg membrane; B, end of body at higher resolving
power; C, with two abdominal segments, enlarged pleuropods (p), retained serosal
sac (s) and minute remains of yolk (y); D, embryo with only one abdominal segment;
in, metathorax (note the enlarged mesonotum and metanotum). I, II, first and second
abdominal segment.
10
V. J. A. NOVAK
successively, one after the other, together with growth of the appendage in
length; short or long mandibles, with two or more teeth, can thus be obtained,
according to the time at which the substance takes effect.
Differentiation of body segments. According to the strobilation theory (cf.
Beklemishev, 1958), the segments of the body are differentiated progressively,
from the anterior towards the posterior end of the body. This was also clearly
demonstrated in a number of cases in which, after application of the test substance at a more advanced stage of embryogenesis (4th-5th day after egg-laying,
Text-fig. 4. Asymmetrical, severely deformed embryo with reduced thorax developing
directly in yolk. Head developing separately from the rest of body without appendages. Crude queen-bee extract.
Text-fig. 5. Severely affected embryos in egg shell. A, Type VI; B, type X, but with
tip of abdomen bent dorsally as at outset of blastokinesis.
room temperature), embryos with a reduced number of abdominal segments
were obtained (Text-fig. 3 A-D), while the thoracic appendages were sometimes
well developed. A curious structure found in one of these specimens (Text-fig.
3C) was undoubtedly the residue of the serosal walls of the original yolk sac.
Asymmetrical specimens were obtained mostly with large doses of strongly
active substances acting in the later stages of development. A very peculiar case
of this type is the specimen in Text-fig. 4, in which the head and the well-differentiated abdomen were found to be separately immersed in the yolk, and the
thorax was not to be found at all. The head had the form of a heart-shaped plate,
with no appendages, but with relatively well-differentiated and pigmented
Juvenile hormone effects
11
compound eyes. The abdomen carried a pair of pleuropods on its rounded
anterior end and laterally a branched segmented appendage was present on the
left side only, between the first and second abdominal segments.
Various stages of blastokinesis were preserved in specimens of types V-VII.
Sometimes, however, well-differentiated type IV or even type II embryos were
found, with the head and thorax in the normal position, but with the abdomen
in the same position as before blastokinesis (Plate 3D; Text-fig. 5A). In these,
blastokinesis was evidently inhibited in about mid-course, while growth of the
Text-fig. 6. Three slightly differing stages of separation of blastoderm from white
cuticle. Hydropylar end of eggs (type XI).
embryo continued. A contrasting case is shown in Text-fig. 5B and partly in
Plate 3B; here the abdomen is bent in the manner typical for type IX, in which
blastokinesis normally does not occur. The only feasible explanation for this
finding is that blastokinesis took place after growth had stopped.
The stage of detachment of the blastoderm from the white cuticle (type XI)
was often combined with a much earlier stage of development (blastoderm with
incipient germinal band formation, the latter usually displaced towards the
anterior end of the egg) than in normal development (end of protopod stage,
beginning of blastokinesis). This could be explained hypothetical ly by assuming
that growth of the embryo stopped, while the serosal moulting process was
initiated at the normal time but was never completed. Several difTerent stages
of detachment of the blastoderm were found (Text-fig. 6,1—III).
The pre-blastoderm stage of the yolk, with surviving cleavage nuclei, was
combined with initial embryogenesis (prior to formation of the blastoderm)
12
V. J. A. NOVAK
(type XII). This is why the white cuticle was not formed in such eggs. In some
cases, however, this state of the yolk was also found in post-blastoderm stages
(types XI and X), together with a small, but more or less differentiated, embryo
with a well-formed white cuticle. The presence of the white cuticle is amply
evident after dechorionation with NaClO. It forms a transparent but tough
B
Text-fig. 7. Giant cleavage cells. A, Cells of various sizes with intervening free space
(consumed yolk). B, Enlarged cleavage cell at high resolving power; note the nucleus,
reduced cytoplasm and three types of inclusions (type XII).
Text-fig. 8. Incomplete transverse bar with circular opening.
Note the highly sclerotic margins.
membrane by which the egg keeps its original form. In its absence, the yolk,
invested only with the thin vitelline membrane, collapses after the chorion has
been dissolved. This stage can be recognized by the coarsely granular appearance
of the yolk. Microscopic examination shows that the reason for this appearance
of the yolk is that it is composed of separate rounded cleavage cells of increased
number and size interspersed with the rest of the yolk mass, while the remaining
space is filled with a transparent fluid. The latter is no doubt the outcome of
consumption of part of the yolk by the cells. The largest cells are 80-100 /*• in
diameter. They probably develop by cryptomitosis. Their cytoplasm contains
Juvenile hormone effects
13
three types of inclusions—large white spheres, small yellow spheres and irregular
transparent granules of gelatinous appearance and intermediate size (Text-fig. 7).
In a number of eggs, specific sclerotic, mainly heavily pigmented bars appeared,
dividing the inside of the egg into two separate compartments. The bars were
often incomplete, leaving a round central opening which sometimes had a more
sclerotic margin (Text-fig. 8). In some eggs, two or more of these bars were
observed. In several cases a small, reduced embryo was found in the largest
compartment. These bars also appeared in eggs attacked by moulds; this seems
to indicate that they developed as a result of cuticulogenic activity of the blastoderm regenerating after annular surface injury by the mould. They appeared
only in embryos severely damaged either by a test substance or by a mould.
TESTS OF VARIOUS JUVENILE HORMONE ANALOGUES
To compare the morphogenetic activity of various substances known for their
juvenile hormone-like effects, eggs of about the same age (1-2 days after laying)
were treated with approximately the same amount of the substance (2-3 microdrops, applied with a thin capillary). The results were evaluated 1-2 days after
hatching of the corresponding controls. The latter were eggs of the same pods,
either untreated or treated with the solvent used in the experiment (acetone,
liquid paraffin, ethanol). Only experiments in which the controls were completely negative were taken into account. The effects of large doses of ethanol
and liquid paraffin were studied separately.
The survey of the results, given in Table 1, shows that the most active test
substances were farnesol methyl ether and other farnesol compounds, such as
dihydrochloromethyl farnesoate (R 24). Crude queen-bee extract had a very
pronounced and specific effect. The synthetic queen inhibitory substance
(9-oxotrans-2-decenoic acid) seems to give less clear results, however, partly
because of the small number of specimens and partly because of the necessity of
applying the substance in ethanol, which appears to have some effect itself.
This question will be studied separately (Novak, 1968). The effect of applying
cecropia oil, whatever its origin, was very slight. The paper factor gave completely negative results. In further experiments farnesol methyl ether was used
almost exclusively, as the most reliable agent (cf. Table 1, no negative results).
Influence of the amount of substance used
To obtain at least an approximate idea of the quantitative aspect of the
effects, eggs of the same age were treated after laying with varying amounts
(different numbers of drops of roughly the same size) of farnesol methyl ether.
The results are given in Table 2.
Even if the number of experiments in some of the series might be thought to
be not altogether adequate, the agreement between the increasing dose and the
3
—
6
4
15
5
2
—
—
n
—
3
5
3
31
10
—
13
1
3
3
3
4
—
4
4
4
7
II
—
4
4
—
—
—
7
—
—
III
2
1
13
3
—
3
2
1
—
IV
4
12
6
3
—
3
—
—
—
V
6
3
—
—
—
2
—
—
VI
—
—
2
—
—
—
—
—
—
VIT
Table 2. Influence ojthe amount ojjuvenilizing substance usea
71
65
19
22
43
35
10
26
0
n
—
—
2
3
6
—
—
—
vi i r
13
—
—
—
2
1
—
—
ix
1
2
6
—
—
3
—
—
—
X
20
10
—
—
2
1
3
—•
XI
7
3
—
4
—
3
2
—
XII
1 drop
2 drops
3 drops
4 drops
5 drops
6 drops
Amount
97
13
38
58
11
53
n
I
10
—
—
—
.—
—
0
20
—
—
—
—
—
9
—
—
—
—
—
II
10
—
5
—
—
3
Hi
14
7
2
1
1
2
IV
4
2
2
8
—
2
V
5
—
—
11
—
—
VI
6
—
10
15
2
8
VII
Type of effect
9
•—
4
—
2
11
VIII
2
2
1
3
—
2
IX
5
—
6
6
4
8
X
13
—
—
2
XI
8
2
4
1
4
6
XII
4
5
6
7
8
9
Mean
(Farnesol methyl ether applied undiluted 0-1 day after egg-laying.) The means were obtained by multiplying the number of
individuals of each type by the corresponding type number (1-XII) and dividing the result by the total number of specimens evaluated
Cecropia oil
Paper factor
Queen-bee extract
9-Oxotrans-2-decenoic acid
9-Hydroxydec-2-enoic acid
10-Hydroxydec-2-enoic acid
R24
Farnesol trans-trans
Farnesol methyl ether
Substance
Type of effect
(In the case of solid substances a particle was placed on the egg surface and was dissolved with a drop of 96 % alcohol.)
Table 1. Comparison oj the effects oj various JH analogues on the embryonic development o/Schistocerca gregaria
(2-3 drops oj undiluted substance per egg)
>
OVAK
Juvenile hormone effects
15
average effect is very clear. This is confirmed by a number of other, less complete,
experiments, which are not given here.
When considering these results the following points must be borne in mind.
The effects of an active substance on the specimen can be modified by three
factors: (i) by its concentration, (ii) by the length of time for which an active
concentration is maintained, (iii) by the interval between application of the
substance to the egg surface and the attainment of an active concentration in a
given structure. With reference to the last point, differences in this respect
between various parts of the embryo could be responsible for the production of
asymmetrical effects if the substance is applied near the crucial period for
determination of a particular morphogenetic process.
Table 3. The influence of time of application
(Farnesol methyl ether, 2-3 drops per egg applied.)
Time of application (h)
24
36
48
62
110
160
182
230
n
51
14
15
.14
14
23
11
14
Type of effect
0
1
—
—
—
—
—
—
—
—
—
2
6
6
3
5
0
8
3
10
8
8
—
2
—
—
—
3
—
—
—
5
4
8
—
2
3
1
—
2
2
—
2
3
2
7
—
1
3
2
1
2
—
2
2
—
1
6
—
2
2
—
1
3
—
1
1
1
3
6
2
2
3
3
2
—
—
—
5
2
4
6
—
—
3
1
—
—
—
—
4
1
2
6
—
2
2
—
2
—
—
—
4
—
4
(7)
II
III
IV
V
VI
VII
VIII
IX
X
XI
XII
Mean
Influence of the time of application
The time of application of the active substance, in relation to the stage of
morphogenesis, is also of primary importance, as well as the amount. This was
tested by subjecting eggs to the action of equal amounts of farnesol methyl
ether (2-3 drops per egg) at different periods after laying. The eggs were kept
at a constant temperature of 32 °C before and after treatment; under these
conditions the untreated controls hatched in about 12 days. The results are
given in Table 3.
Here again, many more experiments, at shorter intervals, would be needed
to obtain an exact idea of the time relations of this type of morphogenetic action.
A detailed analysis of this question is given in Novak (1969 &). The present results,
16
V. J. A. NOVAK
however, like a number of others not given in the table, seem to show a clear
inverse correlation between the activity of the substance and the time at which
it is administered after egg-laying.
CONCLUSIONS
The material studied allows us to conclude that embryonic morphogenesis
can be arrested at any stage by the application of substances with juvenile
hormone-like activity. Development can afterwards continue, under different
conditions, when the substance ceases to act. Any structure characteristic of
embryonic development, starting with the cleavage cells, can thus be kept alive
for a period equal to the hatching time of the control embryos. In addition, a
wide range of new, abnormal structures can appear, either as a result of
heterochronia (in the sense of Novak, 1966) or of direct morphogenetic action.
When comparing juvenilizing effects in embryos with those in larvae, a
striking contradiction can be seen. In post-embryonic development, the administration of juvenile hormone or an analogous substance at a time in development
when it is normally not active (e.g. the beginning of metamorphosis) inhibits
further morphogenesis (metamorphosis) and results in the appearance of giant
larvae. Conversely, removal of the source of the hormone leads to precocious
metamorphosis, resulting in miniature adults. In embryogenesis, however, the
action of a juvenilizing substance quite frequently results in the formation of
fully differentiated dwarf embryos (types III-V).
The following considerations, in terms of the author's gradient-factor theory
(Novak, 1966, 1967), nevertheless suggest that this contradictory effect is not
due principally to a difference in the mode of action of the hormone or analogous
substances, but that it simply arises from the changed physiological and morphological situation, i.e. the different intra-environment (the micro-environment of
Spratt, 1965; cf. Novak, 1967).
It has been shown that miniature embryos appear after the application of
small amounts of the substance to freshly laid eggs. Its effect thus seems to
consist, as in larvae, in causing the survival and further growth of that part of
the embryonic blastoderm which would normally die and disintegrate during
germinal band formation. There is an important difference between this situation
and the situation in the larval period, however. The prolonged growth of larval
tissue necessarily results in the deflexion to larval structures of the part of the
food supply which would otherwise be available for the imaginal structures (cf.
the law of correlation in consumption, Novak, 1966), thus limiting the growth
of imaginal structures.
The situation in early embryos is quite different because of the presence of
an unlimited food supply in the form of the yolk mass. This alone would
adequately explain why further differentiation of the germinal band is not
stopped unless the effect of the JH analogue is strong or persistent. If the sub-
Juvenile hormone effects
17
stance takes effect after differentiation of the germinal band has commenced,
but before it has spread to all the blastoderm cells involved in normal development, its juvenilizing action prevents the differentiation process from spreading
to further cells. When the action of the substance ceases, the embryo would thus
develop from a smaller number of cells than normally; this could be the only
reason for the limited size of juvenilized embryos under the above conditions
and for the associated incomplete consumption of the yolk.
E -b"
rosal moultin
jrmal
5
Eo
•;
v
S
^
*"
5
z a
r
J
IV
VI
VIII
r
IX
Abno
E ;- i
"
XII
:
^
1
2
3
4
5
6
7
8 9
10
Text-fig. 9. Diagrammatic illustration of relation of juvenilized forms to stages of
normal development. On left, different degrees of abnormal development; on right,
stages of normal development; in centre, time (stage) span of some of processes. IV
and XII, proportionate increase in body size caused by juvenilizing factor; IX,
asymmetrical increase.
The same principle also undoubtedly applies at a later stage, in the case of
reduction in the number of body segments (e.g. reduction of the abdomen to
only two metameres). The metameres present at the moment the juvenilizing
substance starts to take effect are preserved and can later differentiate appendages,
but segmentation of the body does not continue. This accounts for the production of embryos with only one or two abdominal segments (Text-fig. 3). The
differentiated segments can continue to grow in their original shape (Text-fig. 9).
Agreement with the juvenilizing effect in the post-embryonic period is clearly
evident here.
More experimental evidence will be needed before all the details observed
can be fully explained. It seems clear, however, that all aberrations produced
by the action of the juvenile hormone and its analogues in embryos could be
caused by the same mechanism as that which produces the changes observed
2
JEEM 21
18
V. J. A. NOVAK
in larval development and metamorphosis and that the differences between
them are due only to the specific intra-environmental conditions and the timing
of action of the substance.
It can be assumed from the above considerations that the main types of
juvenilizing action are as follows.1
(a) Full and persistent action of the juvenilizing factor. This results in the
preservation of structures existing at the moment the factor starts to take effect,
which in normal development would disintegrate. The structures thus preserved
continue to grow as long as the given factor is active and the necessary food
supply (yolk) is available, with the result that they may attain a size several times
larger than normal.
(b) Partial action of the juvenilizing factor, in which only some parts of the
body are more or less juvenilized, while differentiation of the others is not
affected. This is the origin of various forms of heterochronia (cf. Novak, 1956,
1966) found both in the post-embryonic and the embryonic period. These partial
effects have two different causes: (i) the amount of the active substance is too
small to produce a complete effect, (ii) the action of the factor is only temporary,
as a result, for example, of its elimination by some of the tissues. It is impossible
to decide from the available evidence, however, which of these causes applies
in any given case.
(c) Local action of the juvenilizing factor. This may be due either to limited
distribution of the factor, or perhaps more frequently to a low rate of spreading
of the factor inside the body, with the result that different morphogenetic processes are affected, even in symmetrical organs. It can therefore be assumed that
striking changes originate only during the period immediately preceding a determination process. Local action undoubtedly accounts for the various asymmetrical changes observed above.
SUMMARY
1. A number of juvenile hormone analogues and other morphogenetically
active substances (e.g. the queen-bee inhibitory substance) were shown to
produce juvenilizing effects in embryos of Schistocerca gregaria.
2. In about 1500 experimental specimens all transitions were found, from the
initial stages of embryogenesis, starting with preblastoderm cleavage cells
maintained by the juvenilizing effect, to the normal, fully developed embryo.
In addition, other deformities, some of them asymmetrical, were found.
3. To facilitate description of the results, a classification of juvenilizing effects
was suggested, comprising twelve types related to the extent of the morphological changes produced.
1
The term 'juvenilizing factor' covers any factor resulting in juvenilization in the sense
employed above, including the corpus allatum hormone itself and all its analogues, together
with external environmental factors such as temperature (cf. Wigglesworth, 1952) and the
not yet fully understood effect of parasitization by some protozoans (microsporidia) and
helminths (Mermis) (cf. Fischer & Sanborn, 1964).
Juvenile hormone effects
19
4. A number of substances were tested and compared with each other with
reference to the degree of their juvenilizing effects. Farnesol methyl ether and
other farnesol derivatives were found to be the most effective. Distinct specific
activity was also obtained with crude queen bee extract. More evidence is
needed to elucidate the question of the activity of 9-oxodec-2-enoic acid and
some of its derivatives. Cecropia oil and the paper factor (juvabion) of Slama
& Williams had practically no effect in these experiments.
5. The juvenilizing effect was found to increase with the amount of substance
applied and to decrease as the age of the tested eggs rose.
6. Several specific cases of juvenilized locust embryos are described in greater
detail, including fully differentiated miniature embryos, embryos lacking some
of the body segments and surviving giant cleavage cells from the preblastoderm
period.
7. The seeming contradiction between the effects of JH analogues on embryos
(miniature, fully differentiated embryos) and their effects in the post-embryonic
period (giant larvae with inhibited differentiation) can be explained on the basis
of the author's gradient-factor theory as the results of exactly the same mode
of action under different environmental conditions. The fact that embryonic
development can be influenced by juvenilizing factors in the same way as postembryonic morphogenesis is important evidence in support of the gradient-factor
theory.
RESUME
Analyse morphogenetique des effets des analogues de Vhormone juvenile et
d'autres substances morphogenetiquement actives chez les embryons de Schistocerca
gregaria (Forskdl)
1. Un certain nombre d'analogues de l'hormone juvenile ainsi que d'autres
substances morphogenetiquement actives (par exemple la substance inhibitrice
de reine d'abeille) produisent des effets juvenilisants chez les embryons de
Schistocerca gregaria.
2. Chez 1500 specimens experimentaux, on trouve tous les etats intermediaries entre les stades initiaux de l'embryogenese — tel le maintien, du a l'effet
juvenilisant, du stade preblastoderme avec cellules de segmentation — et
l'embryon normal completement developpe. En plus de ces effets, d'autres
deformations, dont certaines sont asymetriques, ont ete observees.
3. Pour faciliter la description des resultats, une classification des effets
juvenilisants est suggeree: 12 stades sont proposes, en fonction du taux de
changements morphologiques produits. Le stade I represente les individus les
moins atteints, le stade XII les individus les plus atteints.
4. Un certain nombre de substances ont ete eprouvees et comparees entre
elles au point de vue de leur effet juvenilisant. L'ether methylique de farnesol
et d'autres derives du farnesol sont les plus efflcaces. Une activite specifique
nette a egalement ete obtenue avec l'extrait brut de reine d'abeille. Des preuves
20
V. J. A. NOVAK
supplementaires sont necessaires pour mettre en evidence l'activite de l'acide
9-oxodec-2-enoique et de certains de ses derives. L'huile de Cecropia et le
facteur extrait de la pate a papier par Slama & Williams (juvabion) sont quasiment inactifs dans ces experiences.
5. L'effet juvenilisant augmente en fonction de la quantite de substance
appliquee et diminue lorsque l'age des oeufs experimentaux augmente.
6. Plusieurs cas speciaux d'embryons d'acridien juvenilises sont decrits avec
plus de detail. Parmi eux, les plus importants sont les embryons parfaitement
differencies mais nains, les embryons depourvus de certains segments du corps,
et les cellules geantes de segmentation, survivantes de la periode preblastoderme.
7. L'examen des resultats obtenus permet de formuler quelques conclusions
importantes sur le mode d'action des facteurs juvenilisants. Les effets apparemment contradictoires produits sur les embryons par des analogues de l'hormone
juvenile (embryons nains completement differencies) et ceux qui sont decrits
comme resultant de Faction des memes facteurs pendant la periode postembryonnaire (larves geantes dont la differentiation est inhibee) peuvent etre
expliques en se basant sur la theorie du facteur gradient proposee par l'auteur:
ces effets resulteraient d'une seule et meme action s'exercant dans des conditions
d'environnement differentes.
8. Le fait que le developpement embryonnaire peut etre influence par des
facteurs juvenilisants de la meme maniere que la morphogenese post-embryonnaire est une preuve importante en faveur de la theorie du facteur gradient.
L'analyse detaillee des phenomenes observes constitue un argument de plus
pour la theorie et ouvre un domaine nouveau d'investigation pour l'etude des
problemes de morphogenese.
Part of the work discussed in this paper was carried out during tenure of a grant from the
Organisation for Economic Co-operation and Development at the time of my stay in the
Anti-Locust Research Centre, London, in the winter of 1965-6. I should like to express my
gratitude to the Director, Dr P. T. Haskell, for providing facilities, for his generous help
during my work at the Centre, and for editing and improving the typescript. Further thanks
are due to Dr D. B. Carlisle, D.Sc, for his interest in my work and for providing technical
equipment and chemicals, and to all members of the staff; to Professor H. A. Schneiderman,
Dr I. G. B. Staal, Dr K. Streibl and Dr K. Slama for providing some of the substances
tested; and to Mrs Margaret Schierlova for corrections to the English of the text.
REFERENCES
BEKLEMISHEV, V. N. (1958). Grundlagen der vergleichenden Anatomie der Wirbellosen. Berlin:
VEB Deutscher Verlag der Wiss. 471 pp.
FISHER, F. M. & SANBORN, R. C. (1964). Nosema, a source of juvenile hormone in parasitised
insects. Biol. Bull. mar. biol. Lab., Woods Hole 126, 235-52.
NOVAK, V. J. A. (1951 a). New aspects of the metamorphosis in insects. Nature, Lond. 167,132.
NOVAK, V. J. A. (19516). The metamorphosis hormones and morphogenesis in Oncopeltus
fasciatus Dal. Acta Soc. zool. csl. pp. 1^48.
NOVAK, V. J. A. (1956). Versuch einer zusammenfassenden Darstellung der postembryonalen
Entwicklung der Insekten. Beitr. Ent. 6, 205-493.
NOVAK, V. J. A. (1966). Insect Hormones, 3rd ed. London: Methuen. 478 pp.
Juvenile hormone effects
21
NOVAK, V. J. A. (1967). The juvenile hormone and the problem of animal morphogenesis.
In Insects and Physiology. Oliver & Boyd, Edinburgh and London, pp. 119-132.
NOVAK, V. J. A. (1969 a). The action of the juvenile hormone as a special type of derepression
activity. Proc. Int. Symp. Insect Endocrin., Brno 1966. (In the Press.)
NOVAK, V. J. A. (19696). Morphogenetical analysis of the effects of the juvenile hormone
analogues on embryos of Schistocerca gregaria. II and III. (In preparation.)
PFLUGFELDER, O. (1947). Die Entwicklung embryonaler Teile von Dixippus morosus in der
Kopfkapsel von Larven und Imagines. Biol. Zbl. 66, 372-87.
RIDDIFORD, L. M. (1966). The action of the juvenile hormone on silkworm embryos. Proc.
Int. Symp. Insect Endocrin., Brno, 1966. (In the Press.)
SLAMA, K. & WILLIAMS, C. M. (1965). Juvenile hormone activity for the bug Pyrrhocoris
apterus. Proc. natn. Acad. Sci. U.S.A. 54, 411-14.
SLAMA, K. & WILLIAMS, C. M. (1966). 'Paper factor' as an inhibitor of the embryonic
development of the European bug Pyrrhocoris apterus. Nature, Lond. 210, 329-330.
SPRATT, N. T. (1965). Introduction to Cell Differentiation. London: Chapman & Hall.
WIGGLESWORTH, V. B. (1952). Endocrine regulation during development. I. Hormones in
relation to metamorphosis. Gen. Comp. Endocrin., suppl. 1, pp. 316-21.
{Manuscript received 20 November 1967, revised 2 August 1968)