/ . Embryol. exp. Morph. Vol. 25, 3, pp. 277-299, 1971
Printed in Great Britain
277
The degree of
determination of the early embryo of Schistocerca
gregaria (Forsk&l) (Orthoptera: Acrididae)
By S. K. MOLOO 1
From the Chelsea College of Science & Technology, London
SUMMARY
The degree of determination of the young embryo of S. gregaria has been investigated
using ligation, thermocautery and centrifugation techniques. From the overall results, it is
suggested that the early development of the embryo is mediated by two physiological centres.
The formation of the germ rudiment is controlled by an activation centre located in the
periplasm round the posterior end of the egg. This centre is already present at the zygote
nucleus stage and is essential during the very early cleavage period.
The differentiation of the germ band is induced by the activity of a second centre, the
differentiation centre, located in the presumptive thorax. It apparently becomes established
at least by the late blastoderm stage and its activity continues during the period of germ-band
formation.
During the late cleavage and early blastoderm stages, the egg is labile and the embryo is
therefore able to normalize its development after part or parts of the germinal Anlage
have been cauterized, removed or displaced.
The differentiation centre completes its functions by the beginning of gastrulation. Thereafter, the embryo is determined.
The embryo can regulate its size at least up to the gastrulation stage provided that a certain
minimum amount of usable yolk is available.
The development of the serosa is not under the control of either centre. This structure seems
to be capable of regeneration providing that a part of the extra-embryonic blastoderm
remains intact.
INTRODUCTION
The early embryonic development of insects is characterized by a co-ordinated
series of morphogenetic events which, in many insects but not all, are mediated
by the activity of physiological centres (Seidel, 1952). Three centres have been
demonstrated to control the beginnings of organization of the embryo: (i) the
cleavage centre which controls cleavage divisions (Krause, 1938, 1957); (ii) the
activation centre which mediates the formation of the germ band (Seidel, 1932;
Reith, 1931, 1935; Ewest, 1937; Brauer & Taylor, 1936; Sander, 1959; Idris,
1960; Mahr, 1960a); and (iii) the differentiation centre which induces differentiation of the germ band (Seidel, 1924, 1936; Reith, 1935; Krause, 1934;
Ewest, 1937; Bock, 1941; Haget, 1953; Idris, 1960; Mahr, 1960/?).
1
Author's address: East African Trypanosomiasis Research Organization, P.O. Box 96,
Tororo, Uganda.
19
E MB 25
278
S. K. MOLOO
Considering the vast number of known insect species, the numbers so far
studied from the point of view of organizer activity is extremely small. Many
more investigations are therefore required before a complete picture of the
mechanisms involved in insect embryonic development can be obtained. The
present paper is concerned with investigations on the organizer activity which
mediates early embryonic development in S. gregaria.
MATERIALS AND METHODS
A culture of S. gregaria, originally provided by the Anti-Locust Research
Centre, was maintained continuously under crowded conditions in an insectarium using the method of Hunter-Jones (1961). The egg pods deposited in
moist sand were developed in an incubator at 28 °C. At this temperature the
eggs hatched 14 days after oviposition.
The normal embryonic development was studied by examination of the
embryos in toto after removal of the chorion in a 3 % solution of sodium
hypochlorite (Slifer, 1945), or after fixation in Carnoy's fluid and dissection
of the outer shell. Some Carnoy-fixed young eggs were sectioned and stained
in Mayer's haemalum/aqueous eosin. Additional information on embryogenesis was obtained from the work by Hong (1968), who used a similar
incubation temperature.
The eggs investigated were those undergoing cleavage (4, 5, 6, 9, 15, 22 and
28 h after oviposition) and at the germ rudiment and gastrulation stages (35
and 45 h, respectively). It is not possible to know the exact state of egg
development at these early stages from external inspection because of the
opacity of the chorion, but all experiments were performed on specimens of
precisely known age. The stages of development corresponding to the ages
at which the experiments were performed are as follows:
Approximate
age (h)
4
5
6
9
15
22
28
35
48
State of development
Zygote nucleus
2 energids
4 energids
8-16 energids
Late cleavage - energids reaching the periplasm at the posterior
end of the egg
Early blastoderm - syncytial blastoderm formed round
posterior half of the egg
Late blastoderm - cellularization of blastoderm completed
Germ rudiment formed round the posterior end of the egg
Gastrulation - inner cellular layer formed and stretches from
the middle of protocephalon to almost the end of protocorm.
Three techniques - ligation, thermocautery and centrifugation - have been
used. For each experimental series a batch of randomly selected eggs was used,
each comprising the contents of one or more pods. The eggs were cleaned
Determination of early locust embryo
279
from attached sand particles and then subjected to one of the three experimental
techniques. About ten eggs from each batch were cultured as controls.
After the operation the eggs were incubated until the sixth day of development.
The chorion of the eggs was then removed in a 3 % solution of sodium hypochlorite and the eggs examined. Those with apparently normal embryos were
allowed to continue development. The remainder were fixed in Carnoy's fluid;
their serosal cuticle, if present, was peeled off under absolute methanol and
the specimens re-examined. Some of these eggs were sectioned and stained in
Mayer's haemalum and counterstained with aqueous eosin.
Ligation technique
The eggs were ligatured by hand using thin nylon thread (diameter, 0 1 mm)
or human hair and both parts of the egg were retained. The operation was
performed under a x 60 binocular microscope. Constrictions were applied at
distances of 5, 35, 50 or 85% of the egg length from the posterior pole.
1 4 cm
A
1-8 cm
i >
Fig. 1. The centrifugation apparatus. (A) for anterior and posterior and (B) for
lateral centrifugation (c.b., centrifuge bucket; c.t., centrifuge tube; g.c, gelatine
capsule; m.l., muslin lining; r.b., rubber buffer; r.t., rubber tube).
Thermocautery technique
An electric thermocautery almost similar to that described by Haget (1953)
was constructed. The instrument had permanent contact electrodes and could
be held conveniently in the hand. Heat was produced with a 6 V battery and
controlled with a switch. The cautery needle consisted of a length of tungsten
wire whose end was ground to a fine point. All operations were carried out
under a binocular microscope at x 60 magnification. The eggs were cauterized
at three different sites: (i) anterior pole, (ii) posterior pole (i.e. the middle of
T9-2
280
S. K. MOLOO
germ Aniage), or (iii) on one side of the extreme posterior end (i.e. at the
periphery of the germ Aniage, referred to as postero-lateral cautery in the text).
Centrifugation technique
An MSE Minor centrifuge used was calibrated for g. In order to maintain
the desired orientation of the specimens during centrifugation the eggs were
placed in gelatine capsules lined with soft muslin to avoid mechanical damage.
The capsules in turn were held in position by means of short pieces of rubber
tubes as shown in Fig. 1. For convenience, Howland's (1941) terminology has
been followed to designate the orientation of the egg axis in relation to the
direction of the force g. Thus, if the posterior pole is centrifugally placed it is
termed posterior centrifugation. In the present study, three orientations for
the eggs were used: posterior, anterior and lateral. Centrifugation periods of
2-5 and 5 min, and four forces, 150, 250, 350 and 500g, were applied.
RESULTS AND DISCUSSION
Approximately 95 % of the total of 680 control eggs, incubated under conditions identical to those of the experimental specimens and obtained from
the same pods, developed normally and hatched.
Table 1. Ligation 5 % of the egg length from the posterior pole
Results
of the
eggs
(h)
4
5
6
9
15
22
28
35
45
(%)
Anterior 95 %
No. of
eggs Z *
Stage of
development
1 nucleus
2 nuclei
4 nuclei
8-16 nuclei
Late cleavage
Early blastoderm
Late blastoderm
Germ rudiment
Gastrulation
46
20
20
59
66
50
69
55
66
4
—
—
10
1
6
6
—
1
Nc
z2
Z3
Z4
Z5
Posterior 5 %
90
65
40
49
35
2
13
22
6
35
60
41
64
92
81
78
19
—
—
—
—
—
—
—
—
80
—\
—
— This portion either
— contained degenerating
— . nuclei or nuclei were
— completely lacking
—
—
—,
Serosa Development
development and
serosal
cuticle
* For explanation of the symbols, see text.
Ligation
The results, expressed as percentages of the total number of eggs ligated at
a particular developmental stage, are given in Tables 1-4 and fall into the
following five categories:
Determination of early locust embryo
281
Burst eggs (Zx). The eggs burst during incubation and some of the yolk was
extruded. These often became infected with bacteria but, whether or not such
infection occurred, the cellular elements degenerated in all eggs.
Table 2. Ligation 35% of the egg length from the posterior pole
Results (%)
Age
f
o
KJl
A
Posterior 35 %
the
eggs
Stage
of
Of
(h)
development
eggs
Zx
30
22
31
20
20
33
20
57
31
—
40
40
48
20
4
9
.15
22
28
35
45
1 nucleus
8-16 nuclei
Late cleavage
Early blastoderm
Late blastoderm
Germ rudiment
Gastrulation
No.
r-
A
z2
Z3
Z4
Z5
Anterior 65 %
33
55
90
40
—
18
—
—
—
—
35
—
40
10
14
10
20
25
34
35
—
—
—
—
—
100% absence of
development. Yolk
abnormally viscous
5
j
Serosa Development
development and
serosal
cuticle
No
Table 3. Ligation 50% of the egg length from the posterior pole
Results (%)
Age
nf
Ul
<
the
eggs
Stage
of
Of
(h)
development
eggs
Zx
z2
28
24
52
32
35
34
20
64
25
8
9
11
38
10
29
—
38
3
11
12
4
9
15
22
28
35
45
1 nucleus
8-16 nuclei
Late cleavage
Early blastoderm
Late blastoderm
Germ rudiment
Gastrulation
No.
A
Posterior 50 %
r-
Z3
;
—
—
—
—
6
30
Z4
7
50
38
9
14
20
20
Z5
Anterior 50 %
25
16
79
64
24
100% absence of
development. Yolk
abnormally viscous
40j
Serosa Development
No
development and
serosal
cuticle
Intact eggs with liquefied or abnormally viscous yolk (Z2). The yolk either
liquefied to a certain extent or became abnormally viscous. These eggs failed
to develop and the nuclei and cytoplasm originally present degenerated.
Eggs in which the serosa and serosal cuticle alone developed (Z3). In these
specimens either a complete or a partial serosa and serosal cuticle developed.
282
S. K. MOLOO
Cell clumps, distributed at random, were observed in some eggs but no development ensued.
Eggs containing abnormal embryos (Z4). The abnormalities observed were:
(a) embryos with gnathos, thorax and abdomen, (b) embryos consisting of
thorax and abdomen, (c) embryos composed of the abdomen alone, and
(d) embryos with protocephalon, gnatho-thorax and with a partial abdomen.
The latter varied in size from a small stump to an almost complete structure.
Table 4. Ligation 85% of the egg length from the posterior pole
Result*5 (%)
Age
the
Stage
eggs
of
(h)
development
4
9
15
22
28
35
45
1 nucleus
8-16 nuclei
Late cleavage
Early blastoderm
Late blastoderm
Germ rudiment
Gastrulation
r
No.
of ,
eggs
30
24
39
20
30
24
30
Posterior 85 %
A
Zt
30
5
18
—
27
8
z2
30
8
21
10
—
12
20
z3
—
—
—
—
—
—
Anterior 15 %
z4
z5
23
29
—
50
6
16
20
58
61 100% absence of
40 development. Yolk
67 abnormally viscous
64
60,
17
1
No
Serosa Development
development and
serosal
cuticle
Dwarf embryos (Z5). Some embryos completed development but were smaller
than normal. The majority of such embryos hatched but their subsequent
fate was not followed.
Specimens showing a complete lack of development (Z7 and Z2) were obtained
after ligation of the eggs at all stages and in all four positions. These results
were almost certainly due to mechanical damage produced by the ligature
and will not be considered further. The results in which development of some
kind occurred in one or the other or in both the regions of the egg are discussed
below.
Ligation at 5 % of the distance from the posterior pole
The results in Table 1 and the observations made are summarized as follows.
(a) Only after ligation at gastrulation did any embryonic development occur
in the anterior portion of the egg; otherwise the serosa alone was formed in
this region. No development of any kind was observed in the posterior section.
(b) The percentage of eggs in which the serosa alone was formed (Z3) was
smallest when the operation was performed at the 1-2 energid and gastrulation
stages, (c) Operation at the gastrulation stage resulted in the development of
abnormal embryos in the anterior section. Such embryos either lacked the
Determination of early locust embryo
283
protocephalon, protocephalon and gnathos or protocephalon and gnathothorax (Z4H_C). In the posterior section the cells degenerated.
At all the cleavage and at the germ rudiment stages constriction isolated
the whole of the presumptive embryonic region in the posterior 5% of the
egg. The cells in the latter portion failed to give rise to an embryo possibly for
three reasons: (i) the constriction damaged the cellular elements and/or periplasm, (ii) there was insufficient yolk and space to permit development, and
(iii) the constriction interfered with organizer activity. Sections showed that
in many cases (i) did in fact occur and, it is conceivable that (ii) is also a likely
explanation. These experiments, however, provide no actual evidence for or
against the third possibility. In the anterior portion, although the cellular serosa
was formed, no embryo developed (Z3). It appears therefore that the posterior
section is essential for embryonic development. The results also indicate that
differentiation of the serosa continues in the absence of the posterior 5% of
the egg. The small number of specimens in which the serosa developed after
the operation at the 1-2 energid stage may be due to the position of the energids
in relation to the ligature. They were either located behind or in the actual region
of the constriction. Only if in the course of the operation the nuclei are pushed
to the anterior side of the ligature would the serosa develop anteriorly. Since
at this stage of development the nuclei are normally posteriorly sited, the small
percentage of eggs in which the serosa was observed is explained.
At the gastrulation stage, in many specimens ligation resulted in abnormal
embryos which developed in the anterior section alone (Z4tt_c). Since by the
time of gastrulation the presumptive embryo normally extends beyond the
site of the constriction, the portions of the germ Anlage isolated anteriorly
developed into those structures which are normally produced by them. It would
appear that the embryo becomes determined at least by the gastrulation stage.
The absence of the protocephalon, protocephalon and gnathos or protocephalon
and gnatho-thorax in the posterior section may be due to one or both of the
following factors: (1) the posterior region is so damaged that no further
development is possible, and (2) the posterior portion is too small to permit
development of these embryonic regions. From the examination of the sectioned
materials it would appear that the first of these explanations is correct.
Ligation at 35% of the distance from the posterior pole
The results presented in Table 2 and the observations made can be summarized
thus: (a) no dwarf (Z5) embryos developed; (b) ligation at all the stages was
followed by the development of some abnormal embryos in the posterior
portion alone; (c) only one type of abnormality was observed, namely the
absence of some of the posterior segments of the abdomen (Z4fZ).
The lack of embryonic development in the anterior 65% of the egg supports
the suggestion made from the previous series of experiments that the posterior
section is essential for embryonic development. There are three possible
284
S. K. MOLOO
explanations for the lack of normal embryos in the posterior 35 % of the egg
and for the type of abnormality observed, (i) The yolk was abnormally viscous,
particularly in the region of the ligature. Hence, the development of that
part of the embryo which would form nearest to the constriction - that is, the
posterior segments of the abdomen - might have been prevented by toxic
substances possibly produced by the altered yolk, (ii) The incomplete embryos
resulted from a shortage of usable yolk. This, however, would not explain
why the posterior segments of the abdomen alone were affected, (iii) The altered
yolk may have interfered with the organizer activity. This is assuming that the
organizer region is located in the presumptive head, thorax or anterior region
of the abdomen, and that the organizer substance diffuses through the yolk.
Chemical changes in the yolk certainly appear to be important, since wherever
defective abdomens were observed, the yolk in the region of the defect was
particularly abnormal. Whether this yolk had a direct effect upon abdominal
development or whether it operated indirectly through hindrance of the diffusion
of organizer substances could not be determined by these experiments.
Ligation at 50% and 85% of the distance from the posterior pole
The results given in Tables 3 and 4 and the observations made show that:
(a) both dwarf (Z5) and incomplete embryos (Z4d) developed in the posterior
section alone; (b) the incidence of dwarf embryos was higher in eggs ligatured
at the 85% than those at 50% level; (c) only one type of abnormality was
observed, namely the absence of some of the posterior segments of the abdomen.
These results, with one exception, are very similar to those obtained from
the previous series of experiments and the same explanations can therefore
be suggested. The exception is the development of some dwarf embryos (Z5)
which did not occur in the previous series. That this should have occurred
and that the number of such embryos should have increased the nearer to the
anterior pole the ligature was applied suggests a simple explanation. That is,
the more space and usable yolk there was, the greater the number of embryos
that managed to complete development, although their size was reduced.
Only one type of abnormality was observed, as before; an absence of the
more posterior segments of the abdomen (Z4(Z). The size of the abdomen varied
from a small stump to an almost complete structure. Furthermore, the embryos
developing in the eggs ligatured closer to the anterior pole generally formed
a larger number of the abdominal segments than those in eggs ligatured at
the 50% level. These results are in accord with the previously suggested
explanations. The farther away the ligature and hence the damaged zone of
the egg is from the developing embryo, the less will its posterior region be
affected by any toxicity of the yolk. Also the farther the ligature from the
future embryo, the more normal will be the yolk in the embryonic region,
particularly near the more anterior regions, and hence the smaller the interference with the diffusion of any possible organizer substances.
Determination of early locust embryo
285
Thermocautery
The results in Tables 5-7 have been grouped into Yj-Y 7 categories described
below.
Burst eggs (Y^. Similar to Z±.
Intact eggs with liquefied or abnormally viscous yolk (Y2). Similar to Z2.
Eggs in which the serosa and serosal cuticle alone developed (Y3). Similar to
Z 3 except that where the serosa enveloped only part of the yolk, the latter
external to the serosa showed an abnormal viscosity and was always found close
to the cauterized region.
Eggs containing abnormal embryos (Y4). The abnormalities observed were:
(a) embryos comprising the thorax and abdomen, (b) embryos consisting of
protocephalon and gnatho-thorax, (c) embryos with protocephalon, gnathos
and abdomen, and (d) embryos with protocephalon, gnatho-thorax and with
partial abdomen. The latter varied in size from a small stump to an almost
complete structure.
Table 5. Cautery at the anterior pole
Age
of the
eggs
Stage
No.
of
Of
(h)
development
eggs
4
5
6
9
15
22
28
35
45
1 nucleus
2 nuclei
4 nuclei
8-16 nuclei
Late cleavage
Early blastoderm
Late blastoderm
Germ rudiment
Gastrulation
80
40
30
63
50
36
60
70
30
Results (% )
r
A
1
—
3
3
8
9
3
—
—
V
•"-2
Y3
Y4
Yr>
Y«
Y7
14
7
—
6
8
22
12
9
10
—
—
—
—
—
—
—
—
—
21
25
17
22
6
14
15
30
27
31
50
50
25
60
33
35
47
33
12
13
10
21
8
—
10
—
7
15
5
20
23
10
22
25
14
23
No
Serosa
development and
serosal
cuticle
Development
i
* For explanation of the symbols, see text.
Eggs containing apparently abnormal embryos (Y5). These embryos apparently
developed normally up to the completion of anatrepsis but in none did katatrepsis occur. All aborted at some stage in the period between these two
embryonic movements.
Dwarf embryos which failed to hatch (Y6). These embryos completed development but were smaller than normal. Such dwarf embryos developed in the
posterior 50, 60 or 70% of the egg, while the anterior portion contained
unusually viscous yolk.
286
S. K. MOLOO
Normal embryos (Y7). Normal embryos developed and in most cases, eclosion
occurred at approximately the same time as the controls.
The Yj. and Y2 eggs were obtained after cautery at each of the three sites, at
all the selected stages. In these specimens it was observed that the burn was
unusually intense and the eggs were either killed immediately or the toxic
substances possibly liberated by the damaged portion diffused to other parts
of the egg so rapidly that the cellular elements were poisoned before any
development could occur. These results will not be considered further.
Table 6. Cautery at the posterior pole
Age
Results I'k 0//Of\
of the
eggs
Stage
of
No.
of
(h)
development
eggs
Yx
Y2
Y3
4
5
6
9
15
22
28
35
45
1 nucleus
2 nuclei
4 nuclei
8-16 nuclei
Late cleavage
Early blastoderm
Late blastoderm
Germ rudiment
Gastrulation
44
30
29
64
60
40
58
62
24
—
—
5
—
3
3
5
—
86
40
76
22
25
20
19
29
37
60
24
73
20
25
78
66
17
A
f
Y4
Y5
Y6
Y7
—
—
—
—
—
—
—
46
—
—
—
36
25
—
—
—
—
—
—
—
—
—
—
—
—
—
—
19
27
—
—
—
14
No
Serosa
development and
serosal
cuticle
Development
Table 7. Cautery at one side of the posterior end
Age
of the
eggs
Stage
of
No.
of
GO
development
eggs
Yi
Y2
Y3
30
24
20
30
30
28
24
25
25
13
—
—
7
—
—
4
—
50
60
45
33
36
32
8
36
4
8
37
40
55
60
10
3
13
—
16
4
5
6
9
15
22
28
35
45
1 nucleus
2 nuclei
4 nuclei
8-16 nuclei
Late cleavage
Early blastoderm
Late blastoderm
Germ rudiment
Gastrulation
Results (
A
V
Y4
—
—
—
—
—
—
—
—
72
Y5
Yc
Y-
—
—
—
—
27
44
75
64
—
—
—
—
—
—
—
—
—
—
—
—
27
21
—
—
—
i
No
Serosa
development and
serosal
cuticle
Development
Determination of early locust embryo
287
Cautery at the anterior pole
The results presented in Table 5 and the observations made show the following.
(a) Abnormal embryos developed after cautery at all the stages; the only
abnormalities observed were a lack of some of the posterior segments of the
abdomen and the altered appearance of the yolk, (b) Eggs containing apparently
normal embryos which aborted at some stage in the period between anatrepsis
and katatrepsis were obtained after cautery at all stages, (c) Dwarf embryos
developed in the posterior region of the egg; anteriorly, the yolk was unusually
viscous; this type of result was obtained after cautery at all except the early
blastoderm and germ rudiment stages, and (d) after cautery at all stages, some
eggs developed normally and hatched.
The results noted in (a) may have been due to toxic substances diffusing
from the burnt region of the egg, which poisoned the yolk together with the
contained vitellophages. Thus, the embryos probably failed to complete development because of a lack of nutrients. This, however, does not explain why only
the posterior part of the embryo was affected. Interference with the differentiation
centre could be responsible. This is assuming that the organizer region is
located in the presumptive head, thorax or the anterior region of the abdomen,
and that the organizer substance diffuses through the yolk. Seidel (1929)
suggests that in Platycnemis pennipes the differentiation centre, located in the
presumptive thorax, does not produce a diffusible substance but acts by
initiating a contraction wave in the yolk system, spreading forwards and backwards, into which the cells of the blastoderm migrate. On the other hand,
Kaulenas (1964) suggests that in Acheta domesticus the organizer substance,
in the form of RNA, spreads progressively posteriorly from the procephalic
differentiation centre. In the present study, the results indicate that wherever
defective abdomens were observed, the yolk in the region of the defect was
abnormally viscous. It is conceivable that diffusion of the organizer substance
from the differentiation centre was affected by this abnormally viscous yolk.
Consequently, the posterior end of the embryo, where the altered yolk was
observed, failed to develop possibly through lack or shortage of the organizer.
This, on present evidence, can only remain a supposition; experiments using
other techniques are needed to confirm or reject it.
At all the stages cauterized, some embryos developed normally up to the
completion of anatrepsis but aborted before katatrepsis (Y5). All such embryos
had the full complement of segments and appeared to be in other respects
normal. In such specimens, the yolk in the anterior portion was unusually
viscous. It is possible that failure to complete development was due to starvation.
In some cases, the embryos completed development but were smaller than
normal (Y6). These dwarfs again developed in approximately the posterior
half of the egg; in the remaining anterior portion the yolk was abnormally
viscous. It appears, therefore, that if a minimum amount of normal yolk is
288
S. K. MOLOO
available, an embryo can complete development although its size is reduced.
If, on the other hand, the amount of usable yolk is below this threshold, the
embryo will abort at some stage before katatrepsis. Thus the embryo can regulate
its size provided a certain minimum amount of normal yolk is available in the
egg. This ability continues at least up to the time of gastrulation. In some
specimens the embryos completed normal development and hatched at approximately the same time as did the controls (Y7), suggesting that the anterior
polar region is not essential for embryonic development.
Cautery at the posterior pole
The results given in Table 6 and the observations made are summarized as
follows, (a) After cautery from the time of the zygote nucleus up to and including
the 8- to 16-nuclei stage, the serosa and serosal cuticle alone developed, (b) After
cautery at the late cleavage and early blastoderm stages, embryos showing
partial and normal development resulted. In the former case, the embryos had
the full complement of segments but aborted at some stage in the period between
anatrepsis and katatrepsis. (c) Neither normal nor abnormal embryos developed
after cautery at the late blastoderm and germ rudiment stages. However, the
serosa and its cuticle were usually present, (d) Abnormal embryos were only
observed after cautery at the gastrulation stage and all exhibited the same
defect: that is, the absence of the thorax.
The results noted in (a) can be explained on the assumption that an activation
centre responsible for the germ-band formation, and located at the posterior
polar region, is already present at the zygote nucleus stage and that it is
essential at least until 8-16 nuclei are present. Hence, cautery of the egg at
the posterior pole during this period of development results in a failure to
form a germ band and hence an embryo. However, as the formation of the
serosa is not inhibited, it would seem that this centre does not control serosal
development. Since in all these specimens the entire yolk appeared normal and
only the periplasm at the site of the burn was damaged, it follows that the
activation centre must be located in the periplasm at the posterior polar
region.
As has been noted above, cautery at the late cleavage and early blastoderm
stages resulted in the development of embryos which either completed development (Y7) or aborted at some stage before katatrepsis (Y5). It appears that
the germ Anlage at those stages were able to regulate the burnt portions since
the embryos exhibited the full complement of segments and seemed otherwise
normal. However, eggs cauterized at the same region at the late-blastoderm
and germ-rudiment stages failed to produce embryos of any kind. Nevertheless,
the serosa and its cuticle developed and the yolk appeared normal.
These combined results can be explained by postulating that embryonic
development is controlled by two organizer centres. The first is the activation
centre, already mentioned, which functions at least during the earliest stages
Determination of early locust embryo
289
of cleavage and is apparently necessary for the formation of the germ band.
If it is assumed that it completes its function at some time during the middle
cleavage period, then if embryonic development occurs after partial destruction
of the original germ rudiment at later stages, the centre is certainly no longer
essential and the egg is evidently labile during this later period. This situation
did in fact obtain during the late cleavage and early blastoderm stages. A centre
of this kind has been demonstrated in P. pennipes (Seidel, 1929), in which
species it acts by producing a diffusible factor. Whether a similar mode of
action is operative in the present species cannot be ascertained on the basis
of present information.
The capacity to form an embryo after posterior cautery might have been
expected to persist until the egg became determined, after which partial embryos
comprising only the undamaged portions would, in suitable circumstances,
have developed. Instead, after cautery at the late blastoderm and germ rudiment
stages, no embryo of any kind was formed. This can be explained by assuming
that at these stages a second centre, the differentiation centre, which mediates
the differentiation of the germ band, is normally operative. If it is assumed
that this centre is determined, then its destruction during the active period
would result in the cessation of embryonic differentiation and ultimately the
abortion of any parts already formed. This is, in fact, what happens after
cautery at the late-blastoderm and germ-rudiment stages. It would therefore
seem that this is the period of activity of the centre. Cautery of the eggs at the
gastrulation stage resulted in partial embryos lacking only the thorax, suggesting
that by this stage the differentiation centre has completed its functions and
the egg is now determined. These eggs were cauterized at the same site as
those at the germ-rudiment stage, and since the gastrulating embryos lacked
the thorax alone, and since the positions of the Anlagen of the major regions
of the body do not alter markedly during the early development period, it
would appear that the differentiation centre is located in the presumptive
thorax.
After cautery at the late cleavage, early blastoderm and gastrulation stages,
the serosa and its cuticle alone developed in some eggs (Y3). In all such specimens
these membranes developed in the anterior region of the egg, while in the
posterior portion the yolk was unusually viscous. Evidently, in these cases
the effects of cautery were so extensive as to prevent any embryonic development.
Cautery at one side of the posterior pole
The results given in Table 7 and the observations made show the following.
(a) After cautery at the early cleavage stages, although the serosa and its
cuticle were formed, no embryo, normal or abnormal, developed, (b) From the
late cleavage to the germ-rudiment stage, some embryos developed exhibiting
the full complement of segments, although, as after posterior-pole cautery, in
many cases the embryo aborted at some stage between anatrepsis and kata-
290
S. K. MOLOO
trepsis. (c) Abnormal embryos developed after cautery at the gastrulation
stage. Two types of abnormalities were observed: (i) embryos comprising the
thorax and abdomen, and (ii) embryos composed of protocephalon and gnathothorax.
A similar type of result to that noted in (a) above was obtained after cautery
at the posterior pole and at the same stages of development. These observations
would appear to support the suggestion that an activation centre is located in
the periplasm round the posterior end of the egg and that the centre is essential
between at least the zygote nucleus and the early cleavage stages. Hence, the
destruction of any part of the periplasm round the posterior end results in
absence of embryonic development.
Postero-lateral cautery from the late cleavage to the germ-rudiment stage
frequently resulted in the formation of embryos with the full complement of
segments (Y5). It is noteworthy that in contrast to the results of posterior-pole
cautery, some embryonic development did occur after the operation was
performed during the late blastoderm and germ rudiment stages. This is the
period during which it was suggested the differentiation centre in the presumptive
thorax was active. The site of cautery possibly explains these results. That is,
the actual differentiation centre was not cauterized, so that the organizer
continued to operate, resulting in embryonic differentiation. The abortion of
these embryos before katatrepsis may be due to a shortage of usable yolk. It was
observed that the yolk in all the Y5 specimens was markedly viscous by the
time that development ceased.
Cautery of the eggs during gastrulation resulted in the development of
embryos which either lacked the protocephalon and gnathos or the abdomen.
These observations support the suggestion that by this stage the embryo is
already determined, the missing embryonic regions being those parts which
are normally produced by the portion of the gastrula cauterized.
Centrifugation
A total of 1404 eggs (450 in the posterior, 491 in the anterior and 463 in the
lateral direction) were centrifuged and their subsequent development followed.
The yolk and cellular elements of all the eggs subjected to centrifugal forces
stratified into three zones. From the centripetal pole, these were: (i) a yellow
layer of large yolk globules occupying about 30 % of the egg, (ii) a narrow
layer composed of protoplasmic elements, and (iii) an extensive zone of granular
yolk. This arrangement conforms qualitatively to that of the centrifuged eggs of
Musca domestica (Pauli, 1927) and of Chironomus dorsails (Yajima, 1960).
Since normally the protoplasmic elements at the stages of development studied
are predominantly located in the posterior quarter of the egg, these components
are shifted to a greater distance by posterior than anterior centrifugation.
At all the stages of development selected for the investigation, the lowest
centrifugal force used for the shortest time - that is 150g for 2-5 min - produced
Determination of early locust embryo
291
stratifications which merged into each other at their boundaries. As the gravitational force and length of time were increased, the boundaries between the
zones became more clearly demarcated.
There was a tendency for the stratification to break down after centrifugation
at lower speeds had ceased. After exposure to \50g the constituents of the
yolk became redistributed after about 3 h, while after stronger forces the
process was completed after a much longer period, and in many eggs, particularly
after the exposure to forces greater than 250g, the stratification remained
permanent. Furthermore, after lateral centrifugation, the redistribution of the
egg contents was accomplished more quickly than in specimens operated upon
in the other planes.
The results obtained fall into three broad groups; those in which no development occurred, those in which the serosa and its cuticle alone developed, and
those in which embryos, both normal and abnormal, developed. The abnormalities observed were: (1) embryos comprising the protocephalon alone,
(2) embryos consisting of the protocephalon and gnathos, (3) embryos composed
of the thorax alone, (4) embryos comprising the gnatho-thorax, (5) embryos
consisting of the protocephalon and gnatho-thorax, (6) embryos comprising
the protocephalon, gnatho-thorax and a partial abdomen varying from a small
stump to an almost completely formed structure, (7) complete embryos which
were abnormally orientated, and (8) dwarf embryos in some of which the
appendages were abnormally twisted and/or some of the yolk was left outside
the embryo at dorsal closure (these dwarfs failed to eclode).
Abnormalities of the types (1) and (2) were observed only after treatment of
the eggs at the gastrulation stage; types (3) and (4) after treatment at the late
blastoderm and germ-rudiment stages, and types (5) and (6), in general, after
operation at the germ-rudiment stage. Types (1) and (8) were observed in some
of the eggs treated at all except the 1-2 energid and early cleavage stages.
The three groups of results are presented graphically in Figs. 2-5.
(i) Eggs in which no development occurred
The results showing complete lack of development (Fig. 2) are summarized
thus: (a) the number of eggs in which development failed to occur was generally
highest after posterior centrifugation and lowest after lateral centrifugation;
(b) the number increased with the increase in the centrifugal force irrespective
of the plane of treatment; (c) the number was particularly high after treatment
at the 1-2 energid, early cleavage, late blastoderm and germ-rudiment stages.
In some specimens the complete lack of development was possibly due to
the cellular damage caused by the operation. The cellular elements at the
stages of development investigated are predominantly located in the posterior
region of the egg. Centrifugation pushes these components through the viscous
yolk, possibly causing damage in some specimens. Irrespective of the duration
of operation, posterior centrifugation shifts the cellular elements through the
292
S. K. MOLOO
1-2E EC LC EB LB GB GN 1-2E EC LC EB LB GB GN 1-2E EC LC EB LB GB GN 1-2E EC LC EB LB GB GN
g = 15O
g = 25O
g = 35O
g = 500
Fig. 2. The incidence of total lack of development after centrifugation ait various
speeds, expressed as a percentage of all the results. E, Energids; EC, early cleavage;
LC, late cleavage; EB, early blastoderm; LB, late blastoderm; GB, germ band;
GN, gastrulation.
ra
u
1-2E EC LC EB LB GB GN 1-2E EC LC EB LB GB GN 1-2E EC LC EB LB GB GN 1-2E EC LC EB LB GB GN
g=150
g = 25O
g = 35O
g = 500
Fig. 3. The incidence of embryonic development after centrifugation at various
speeds, expressed as a percentage of all the results. Abbreviations as in Fig. 2.
Determination of early locust embryo
293
1-2E EC LC EB LB GB GN 1-2E EC LC EB'l_B GB GN 1-2E EC LC EB LB GB GN 1-2E EC LC EB LB GB GN
g = 150
g = 250
g = 350
g = 500
Fig. 4. The incidence of complete (dark) and partial (plain) embryos after centrifugation at various speeds, expressed as a percentage of the number of specimens
showing embryonic development. Abbreviations as in Fig. 2.
100
75
50
Illllll .
25
0
100
75
50
I4U I ll
100 r
75
Hi
1-2E EC LC EB L B G B G N 1-2E EC LC EB L B G B G N 1-2E EC LC EB L B G B G N 1-2E':EC LC EBLBGBGN
g = 150
g = 250
g = 35O
g = 500
Fig. 5. The incidence of development of the serosa and its cuticle alone after
centrifugation at various speeds, expressed as a percentage of all the results.
Abbreviations as in Fig. 2.
E M B 25
294
S. K. MOLOO
greatest distance, while treatment in the lateral direction shifts them through
the least. Consequently, the incidence of the mechanical damage would be
expected to be highest in the case of the former. Further, should the relative
gravitational force be stepped up, the rate of movement of protoplasm would
be expected to increase, thus resulting in a higher incidence of disruption. The
results noted in (a) and (b) above could therefore be explained, at least in
part, in terms of the disruption of energids and cells. It is also possible that the
stratification impedes migration of intact cellular elements and that the hindrance
to such migration is probably greater after posterior than anterior centrifugation,
since after treatment in the former direction the heavy yolk was distributed
to the posterior region where the embryo normally begins to develop, while
after anterior centrifugation the posterior region of the egg contained lighter
yolk. Treatment in the lateral direction shifted the cellular elements a very
short distance and, since the yolk is laterally stratified, their normal migration
was almost certainly least affected. The lack of development could also therefore
be explained, at least in some specimens, in terms of the impedance to cellular
migration resulting from yolk stratification. It seems highly probable that both
factors may operate, sometime together, to inhibit development.
The results of the thermocautery experiments suggested that the activation
and differentiation centres are operative during the very early cleavage period
and the late blastoderm and germ-rudiment stages, respectively. The eggs would
therefore be expected to be particularly sensitive to centrifugation during
these periods. That this is, in fact, correct is evident from Fig. 2 which shows
that a markedly larger number of eggs failed to develop after treatment at
these stages than at any other period. Treatment at 150g at the 1-2 energid and
early cleavage stages was followed by redistribution of the egg constituents, a
process which took about 3 h to accomplish. It is possible that the stratification
obtained prior to the completion of this process affected the activation centre,
possibly through hindrance of the organizer diffusion. The embryo is labile
during the late cleavage and early blastoderm stages, so that after the operation
during these periods of development, regulation was possible. Centrifugation
of the specimens at the late blastoderm and germ-rudiment stages, during which
the differentiation centre is operative, gave results in which a significantly
large number failed to develop. It is conceivable that this centre was affected
in a similar manner to the activation centre and that this led to the death of
the eggs. The incidence of complete lack of development was markedly low
after treatment at gastrulation. This result can be explained by assuming that
the cells of the gastrulating embryo are more tightly bound together than those
of the earlier stages, so that less cellular disruption is caused by the treatment.
(ii) Eggs in which embryos, both normal and abnormal, developed
The results showing embryonic development of some kind (Figs. 3, 4) can
be summarized as follows, (a) The number of eggs in which embryos developed
Determination of early locust embryo
295
was generally low after treatment at the 1-2 energid, early cleavage, late
blastoderm and germ-rudiment stages, (b) A relatively larger number of
embryos continued to develop after centrifugation at the late cleavage, early
blastoderm and gastrulation stages, (c) After centrifugation at all except the
1-2 energid and early cleavage stages, some partial embryos developed. The
incidence of abnormalities was particularly high after treatment at the late
blastoderm and germ-rudiment stages.
If the conclusions drawn from the thermocautery results are correct, then
it would be expected that after treatment during the very early cleavage period,
none or least only a small number of specimens would continue to develop.
This was in fact the case. Evidently in a few specimens in which development
did occur, the activity of the activation centre was in no way affected by the
treatment. Possibly the redistribution of the egg constituents was accomplished
unusually rapidly and thus the organizer activity was unaffected. At the late
cleavage and early blastoderm stages the egg is labile and the embryo therefore
managed to 'regulate' after the operation. In fact in the majority of the
specimens the embryos had the full complement of segments, though, in a few
cases, these were abnormally orientated. The incidence of embryonic development was low after treatment at the late blastoderm and germ-rudiment stages.
This lends further support to the suggestion that the differentiation centre is
operative during this period. In many specimens only partial embryos developed
but in all such eggs the thorax was formed. This suggests that the differentiation
centre is located in the presumptive thorax and supports a similar suggestion
made to explain the thermocautery results.
As was expected, a relatively large number of eggs continued to develop
after centrifugation during gastrulation. Although the majority of embryos
had the full complement of segments, a few partial embryos were formed.
The only explanation that can be offered for those cases in which incomplete
embryos developed is that the treatment so violently dislocated and damaged
a part of the determined embryo that this region aborted.
(iii) Eggs in which the serosa, but no embryo, developed
The results presented in Fig. 5 indicate that in some specimens formation
of the serosa and its cuticle occurred after the operation, although no embryo
developed. The incidence of this type of result was very sporadic. Since the
serosa was differentiated in eggs in which the activation centre was rendered
ineffective by the operation, it would seem that this centre does not control
serosal development. In those specimens in which this type of result was
obtained after centrifugation at the later stages of development, it is possible
that the germ rudiment whose development is mediated by the organizer was so
damaged by the centrifugal force that no embryonic development was feasible.
In some cases, although the operation disrupted at least part of the extraembryonic blastoderm, the serosa was formed. It seems therefore that, provided
296
S. K. MOLOO
a portion of the extra-embryonic blastoderm remains intact, it can regenerate
a part or the whole of the missing portion.
DISCUSSION
The early embryonic development of S. gregaria appears to be controlled
by at least two organizer centres: the activation and differentiation centres.
The cleavage centre which controls cleavage divisions in Tachycines asynamorus
(Krause, 1938) and Notonecta glauca (Krause, 1957) could not be detected in
the present study.
The periplasm round the extreme posterior end, which has been termed
the activation centre, mediates the formation of the germ band and hence
an embryo. This centre is present at the zygote nucleus stage and is essential
up to the time when 8-16 nuclei are present. Hence, amputation of this region
during this period of development results in absence of embryonic development.
In Platycnemis pennipes (Seidel, 1929) this centre produces a diffusible substance
after being activated by the passage into it of cleavage nuclei. Whether this
mode of action is operative also in S. gregaria could not be determined
directly, but the results obtained seem to point to the presence of a diffusible
factor.
Haget (1953), who studied the embryonic organizer system in Leptinotarsa
decemlineata, refutes the presence of such a centre in any insect egg and
interprets Seidel's (1929) findings as due to the inability of P. pennipes egg to
tolerate any mechanical damage at its posterior end. His condemnation of the
concept of an activation centre is unfounded. A centre of this type has been
demonstrated in many other insects; for example, Camponotus ligniperda
(Reith, 1931), Tenebrio molitor (Ewest, 1937), Bruchus quadrimaculatus (Brauer
& Taylor, 1936), Sitona lineata (Reith, 1935), Euscelis plebejea (Sander, 1959),
Culexfatigans (Idris, 1960) and Acheta domesticus (Mahr, 1960a). Without this
centre embryonic development does not occur. The fact that such a centre has
not been detected in some other insects does not necessarily disprove its presence
in the above examples. It is conceivable that it operates in different ways in
different insect species. For example, in contrast to P. pennipes (Seidel, 1929),
development of the germ band in E. plebejea (Sander, 1959) and Chironomus
dorsalis (Yajima, 1964) is mediated through interaction between anterior and
posterior factors. It may be that in still other species a more widespread region
of the egg is concerned.
Amputation of the posterior end during the late cleavage and early blastoderm
stages did not inhibit embryonic development. It would seem that the activation
centre is no longer essential after the 8- to 16-nuclei stage and thus the embryo
is thereafter labile. The embryo is therefore able to normalize its development
after part or parts of the germinal Anlage have been cauterized, removed or
displaced.
Determination of early locust embryo
297
Cautery of the presumptive thorax during the late blastoderm and germ-band
stages resulted in a lack of embryonic development, whereas, after the destruction of other regions of the germ Anlage during the same developmental stages,
embryonic differentiation continued. It would seem that during this period of
development a second centre, the differentiation centre, which is located in
the presumptive thorax and which induces differentiation of the embryo, is
operative. The results of centrifugation experiments have supported these
suggestions. Although there is no direct evidence to suggest that this centre
acts by giving out a diffusible substance, the results seem to give some indication
of the presence of such a factor.
After this second centre has completed its functions - that is, by the gastrulation stage - the prospective embryo becomes determined. Hence, destruction
or removal of any region of the gastrula resulted in the development of an
embryo lacking in those structures which are normally produced by the region
eliminated.
After treatment using all the three techniques, dwarf embryos developed in
some specimens. Such embryos developed in approximately the posterior half
or more of the egg; in the remaining anterior portion the yolk was abnormally
viscous. It therefore appears that the embryo can regulate its size provided
a certain minimum amount of nutrients is available in the egg.
Since the differentiation of the extra-embryonic blastoderm into serosa
continued in many specimens in which the influence of the activation and the
differentiation centres were eliminated by the treatments, it appears that the
development of the serosa is not controlled by either centre. This structure
seems to be capable of regeneration provided that a part of the extra-embryonic
blastoderm remains intact.
RESUME
Le degre de determination dujeune embryon de Schistocerca gregaria
(Forskdl)
On a etudie le degre de determination dujeune embryon de S. gregaria par des techniques
de ligature, de cauterisation thermique et de centrifugation. De Pensemble des resultats, il
apparait que le debut du developpement embryonnaire est regi par deux centres physiologiques.
La formation de Pebauche embryonnaire est controlee par un centre d'activation localise
dans le periplasme a la partie posterieure de l'ceuf. Ce centre est deja present au stade zygote
(du noyau) et est essentiel durant le tout debut de la periode de segmentation.
La differenciation de la rondelette germinative est induite par l'activite d'un second centre,
le centre de differenciation, localise dans le thorax presomptif. Ce centre semble s'etablir au
stade blastoderme avance au moins et sur activite continue durant la periode de formation
de la bandelette germinative.
Pendant la fin de la periode de segmentation et au debut du stade blastoderme, l'ceuf est
labile et de ce fait, l'embryon est capable de normaliser son developpement apres qu'une
partie ou des parties du territoire aient ete cauterisees, enlevees ou deplacees.
Le centre de differenciation termine ses fonctions au debut de la gastrulation. Apres ce
stade, l'embryon est determine.
L'embryon peut effectuer la regulation de sa taille au moins jusqu'au stade gastrula pour
autant qu'une certaine quantite minimum de vitellus utilisable soit disponible.
298
S. K. MOLOO
Le developpement de la serose n'est pas sous le controle de ces deux centres. Cette
structure semble capable de regeneration pour autant qu'une partie du blastoderme extraembryonnaire reste intacte.
This work is part of a thesis submitted to the University of London for the degree of
Ph.D. I wish to thank Dr M. F. Sutton for supervision, Dr F. K. Dar for reading the
manuscript and the governors of the college for a research grant.
REFERENCES
BOCK, E. (1941). Wechselbeziehungen zwischen den Keimblattern bei der Organbildung von
Chrysopaperla (L.). I. Die Entwicklung des Ektoderms in Mesodermdefekten Keimteilen.
Wilhelm Roux Arch. EntwMech. Org. 141, 159-247.
BRAUER, A. & TAYLOR, A. C. (1936). Experiments to determine the time and method of
organization in Bruchid (Coleoptera) eggs. /. exp. Zool. 73, 127-152.
EWEST, A. (1937). Struktur und erste Differenzierung im Ei des Mehlkafers Tenebrio molitor.
Wilhelm Roux Arch. EntwMech. Org. 135, 689-752.
HAGET, A. (1953). Analyse experimentale des facteurs de la morphogenese embryonnaire chez
le Coleoptere Leptinotarsa. Bull. biol. Fr. Belg. 87, 123-217.
HONG, C. (1968). Descriptive and Experimental Studies on the Embryonic Development of
Schistocerca gregaria. Ph.D. thesis, University of London.
HOWLAND, R. B. (1941). Structure and development of centrifuged eggs of Drosophila
melanogaster. Proc. Am. phil. Soc. 84, 605-616.
HUNTER-JONES, P. (J961). Instructions for Rearing and Breeding Locusts in the Laboratory.
London: Anti-Locust Research Centre.
IDRIS, B. E. M. (1960). Activation centre in Culex. Wilhelm Roux Arch. EntwMech. Org.
152, 230.
KAULENAS, M. S. (1964). General and Experimental Embryology o/Achetad omesticus (£.).
Ph.D. thesis, London University.
KRAUSE, G. (1934). Analyse erster Differenzierungsprozesse im Keim der Gewachshausheuschrecke durch kunstlich erzeugte Zwillings-Doppel und Mehrfachbildungen. Wilhelm
Roux Arch. EntwMech. Org. 132, 115-205.
KRAUSE, G. (1938). Die Ausbildung der Korpergrundgestalt im Ei der Gewachshauscheuschrecke Tachycines asynamorus. Z. Morph. Okol. Tiere 34, 499-564.
KRAUSE, G. (1957). Neue Beitrage zur Entwicklungsphysiologie des Insektenkeimes. Verh.
dt. zool. Ges. (Graz), 396-424.
MAHR, E. (1960tf). Normale Entwicklung, Pseudofurchung und die Bedeutung des Furchungszentrums im Ei des Heimchens (Gryllus domesticus). Z. Morph. Okol. Tiere 49,
263-311.
MAHR, E. (19606). Struktur und Entwicklungsfunktion des Dotter-Entoplasmasystems im
Ei des Heimchens (Gryllus domesticus). Wilhelm Roux Arch. EntwMech. Org. 152, 263—
302.
PAULI, M. E. (1927). Die Entwicklung geschniirter und centrifugierter Eier von Calliphora
erythrocephala und Musca domestica. Z. wiss. Zool. 129, 483-540.
REITH,F. (1931). Versuche iiber die Determination der Keimanlage bei Camponotus ligniperda.
Z. wiss. Zool. 139, 664-734.
REITH, F. (1935). Ober die Determination der Keimesanlage bei Insekten (Ausschaltversuche
am Ei des Riisselkafers Sitona lineata). Z. wiss. Zool. 146, 77-100.
SANDER, K. L. (1959). Analyse des Ooplasmatischen Reaktionsystems von Euscelis plebejus
durch Isolieren und Kombinieren von Keimteilen. I. Die Differenzierungsleistungen
vorderer und hinterer Eiteile. Wilhelm Roux Arch. EntwMech. Org. 151, 430-497.
SEIDEL, F. (1924). Die Geschlechtsorgane in der embryonalen Entwicklung von Pyrrhocoris
apterus. Z. Morph. Okol. Tiere 1, 429-506.
SEIDEL, F. (1929). Untersuchungen iiber das Bildungsprinzip der Keimanlage im Ei der
Libelle Platycnemis pennipes I-V. Wilhelm Roux Arch. EntwMech. Org. 119, 322-440.
Determination of early locust embryo
299
F. (1932). Die Potenzen der Furchungskerne im Libellenei und ihre Rolle bei der
Aktivierung des Bildungszentrums. Wilhelm Roux Arch. EntwMech. Org. 126, 213-276.
SEIDEL, F. (1936). Entwicklungsphysiologie des Insektenkeims. Verh. dt. zool. Ges. 38,
291-336.
SEIDEL, F. (1952). Entwicklungsphysiologie des Insekteneies. Fortschr. Zool. 9, 620-699.
SLIFER, E. H. (1945). Removing the shell from living grasshopper eggs. Science, N.Y. 102,
282.
YAJIMA, H. (1960). Studies on embryonic determination of the harlequin-fly, Chironomus
dorsalis. I. Effects of centrifugation and its combination with constriction and puncturing.
/. Embryol. exp. Morph. 8, 198-215.
YAJIMA, H. (1964) IF. Effects of partial irradiation of the egg by ultra-violet light. /. Embryol.
exp. Morph. 12, 89-100.
SEIDEL,
{Manuscript received 28 April 1970)