/ . Embryo/, exp. Morph. Vol. 23, 3, pp. 705-718, 1970
705
Printed in Great Britain
Quantitative estimation of the stage of embryonic
development in the locust, Schistocerca gregaria
ByN. M. T Y R E R 1
From the Department of Zoology, Cambridge University, England
An understanding of the physiological and structural development of an
embryo requires a knowledge of the precise state that development has reached.
Without this knowledge it is not possible to compare findings from experiments
performed on different embryos or to relate them to structural changes during
development.
In previous work on Acridid embryos two methods have been used in estimating the stage of development. Some authors have defined embryonic stages
by their age from the time of oviposition (Slifer, 1932; Roonwal, 1936, 1937;
Hong, 1968), while others have described development in a series of arbitrarily
chosen stages, which are based primarily on changes in the external morphology
(Steele, 1941; Jhingran, 1947; Mathée, 1951; Shulov & Pener, 1959, 1963).
The eggs of Schistocerca gregaria are laid in pods of 30-90. Pods were collected
and kept under constant conditions in order to investigate the development of
the embryonic muscle (Tyrer, 1968, 1969). During this work it was found that
there was considerable variation in the differentiation of eggs from different pods
of the same age, even though most of the eggs in any one pod were at the same
stage. This is reflected by the length of the development period of the eggs, which
at a temperature of 28 °C, ranged from 13 to 20 days with a mean of 15-25 days
(Fig. 1). Similar variations have also been observed by other workers on Acridid
embryos (Slifer, 1932; Shulov & Pener, 1959, 1963). Because of these large
variations, time alone cannot be used to define the stage of development accurately, especially in the later stages where the effects of variation will be more
marked.
This difficulty can be avoided by defining development in a series of stages
based on the changes in external morphology, but this has the disadvantage that
internal changes may take place without changes in external features, and may
therefore occur in the undefined period between stages. Shulov & Pener's series
for S. gregaria (1963) defines the early stages in detail and it is these which are
the main concern of the embryologist, interested in the origin and formation of
organs. The physiologist, however, is concerned primarily with the function of
1
Author's address: Department of Biology, Gilmer Hall, University of Virginia, Charlottesville, Virginia 22903, U.S.A.
706
N. M. T Y R E R
organs which develops towards the end of embryonic life. In this period most of
the external changes are slight and often qualitative (e.g. progressive pigmentation), and Shulov & Pener give few stages which are difficult to identify precisely.
Rather than the discontinuous steps of a morphological series, investigation of
organ function requires a continuous scale of development such as that provided
by timing, but one which can be adjusted to the differing rates of development.
A method has been devised, therefore, which enables any point in development to be defined accurately, by expressing the age of an embryo as a percentage
of the development time of the rest of the eggs in the pod. The validity of the
320 330 340 350 360 370 380 390 400 410 420
Time (h)
Fig. 1. The time from oviposition to hatching in 75 egg pods kept at 28 °C.
method has been checked by correlating external changes in the later stages with
percentage development calculated from accurate measurements of the incubation period. From the data obtained it has been possible also to define stages
for the later part of development which can be used when the more accurate
method is not required, and which are more precise than those given by Shulov
& Pener (1963).
METHODS USED TO MEASURE THE INCUBATION PERIOD
A large stock of Schistocerca gregaria is kept in the Zoology Department,
Cambridge, using methods similar to those of the Anti-Locust Research Centre
(Hunter-Jones, 1961). 50-60 adults were kept in a standard, glass-fronted culture cage 18" x 18" x 20", in a constant temperature room maintained at 28 ± 1 °C
and a relative humidity of 35-40 %.
To record the time of oviposition
The pods of eggs of Schistocerca are normally laid in moist soil. The tubes of
wet sand usually provided for oviposition (Hunter-Jones, 1961) were replaced by a
trough 24" x 4£" x f "filledwith wet sand. This was drawn on a trolley below the
Stage of embryonic development
707
culture cage at a rate of i"/h by a geared-down electric meter motor fixed to the
cross-bar between the rails at the end of the trolley's run (Fig. 2). The locust
had access to it via a IV' diameter hole cut in the floor of the cage. According
to the position of the pod in the trough the time of oviposition could be determined to within 2 or 3 h. The females seemed undisturbed by having to lay in a
narrow, moving trough. Initially a normal, stationary tube was provided in
addition to the trough, and it was found that pods were laid in both receptacles
with equal frequency.
Cage
Laying hole
Collecting trough
Trolley
Rails
Pulley
Electric motor
Counter weight.
Fig. 2. A p p a r a t u s for recording the time of egg laying.
The trough was removed once every 48 h, and the individual egg pods were
transferred with some of the sand surrounding them to small covered glass jars
on which the time and date of oviposition was written. The jars were placed in
polythene bags to prevent evaporation from the wet sand.
To determine the time of hatching
A single pod of eggs was placed in apparatus which recorded the emergence of
the first hopper as it crossed a light beam (Fig. 3). The resulting change in light
intensity on a photocell switched on a transistor operating a relay which actuated
a lever writing on a smoked drum. The lid of the jar containing the eggs was
removed and replaced by a cover incorporating a short, vertical tube | " in
diameter, which allowed the newly hatched hopper to enter the recording apparatus. This consisted of a Tufnol tube, Y in diameter, mounted vertically and
leading to a collecting jar. Half-way up the tube a narrow horizontal beam of
708
N. M. T Y R E R
light from a 6 V, 0-3 A torch bulb was directed, by means of a wave-guide made
from a specially shaped piece of Perspex, across the tube on to a photoconductive
cell (Mullard ORP 12) mounted in the opposite wall. The whole apparatus was
kept in an incubator maintained at 28 ±0-5 °C.
A disadvantage of this apparatus is that it does not give a record of the variation in hatching times of the eggs in the pod since the hatchlings tend to congregate around the light so that only the time that the first one emerged can be
measured. This could perhaps be overcome by using a red light beam to which
the hoppers are not attracted and by providing a white light in the collecting jar
to attract them.
•
8-2 K
Collecting flask
-12V
160 Ci
Relay operating
pen recorder
ORP 12
-. +12V
'Tufnol ' tube
Photoconductive cell
Diagrammatic section through light system
Waveguide
'Tufnol'tube
Wave guide
6 V lamp
Photoconductive
cell (ORP 12)
Fig. 3. Apparatus for recording the time of hatching of eggs.
CORRELATION OF PERCENTAGE AGE WITH DEVELOPMENT
Expressing the age as a percentage of the development time is a precise
definition of the stage that the embryo has reached only if two criteria are
fulfilled. First, all the embryos in one pod must develop at the same rate, and
second, the development of each embryo must proceed at a constant rate.
(J) Synchronous development in the pod
Salzen (I960) found in Locusta migratoria migratorioides that the eggs in any
one pod were usually at the same stage of development. To check that this is
also true for Schistocerca,fiveeggs were removed from each of 50 pods. The egg
shell was dissected away under the binocular microscope and the external features of the embryos compared. Usually all five embryos were morphologically
identical although occasionally there were some retarded individuals which were
Stage of embryonic development
709
usually abnormal and were discarded. Papillon (1960) described pods laid by the
' gregaria' phase of S. gregaria containing a small percentage of ' solitaria' forms
which occurred in the lower third of the pod. She found that these eggs hatched
ca. 20 h later than the 'gregaria' at 30 °C. Although this phenomenon was not
observed here, differences in phase character were obviated by using eggs only
from the upper two-thirds of the pod.
Further evidence that all the embryos in one pod develop at the same rate
comes from measurement of the variation in their development time. Although
the hatching recorder did not give a record of this variation (see above) this was
checked in 12 pods by removing the hatchlings from the apparatus at hourly
intervals and counting them (Table 1). These results show that 49% of the eggs
hatch within 2 h of the first hatchling and 82% have hatched by 3 h.
Table 1. Distribution of the numbers of individuals hatching
from single pods
Number of hours after appearance of first hatchling
A
Pod
1
2
3
4
5
6
7
8
9
10
11
12
Total
1
2
3
10
3
2
3
26
5
1
12
7
39
2
7
117
15
19
10
7
0
10
7
0
12
2
10
15
107
9
7
10
50
7
25
3
12
17
5
0
7
152
12
0
0
3
25
3
0
7
15
14
5
5
0
77
Total
24
0
0
0
1
0
1
0
1
0
0
0
3
6
459
Each column gives the number of hoppers which hatched iin the period since the previous
reading.
(2) Constant rate of development of individuals
It is generally accepted that eggs of tropical locusts such as Schistocerca
develop continuously (Lees, 1955; Shulov & Pener, 1961). Development is
arrested only if the soil in which the eggs are laid dries up, and is resumed when
the soil is moistened again (Shulov & Pener, 1963). In this study, particular care
was taken to prevent drying of the eggs (see above) to avoid this. Even so, it is
possible that the rate of development of an embryo could fluctuate during
incubation, and that embryos from different pods could have different fluctuations in the rate of development. This was excluded by an analysis of the external
features of embryos at the later stages of development.
710
N. M. T Y R E R
Six egg pods between 9-5 days (227 h) and 13-5 days (323 h) old were chosen
for a detailed analysis. They were kept in an incubator at 28 ± 0-5 °C. Five eggs
were taken from each pod every day at exactly 24 h intervals. These were dissected in Hoyle Ringer (Hoyle, 1953) and the embryos examined alive under the
binocular microscope. The changes which occurred in 16 characters were noted
(Table 2), and the animals fixed in neutral formol saline. The remaining eggs in
Table 2. Characters assessed in the morphological analysis of the
later stages of development
Character
1
2
3
4
5
6
7
8
9
10
11
12
Ratio of length of metathoracic femur to side-to-side body
width
Crescent present on metathoracic femur
Femur crescent pigmented
Spines on metathoracic tibia
Tibial spines pigmented
Pigment present on tip of metathoracic tarsus
Mandible differentiated into teeth
Mandibular teeth pigmented
Opacity; yolk visible through dorsolateral body wall
Yolk visible through legs
Pigment spots present on legs (and abdomen)
Lighter than yolk
Same shade as yolk
Darker than yolk
Pigment spots on thorax and head
13 Hairs present on the antenna
14 Presence of pigment spot on the cercus
Ratio of width of pigmented area of the eye, P, to width of
non-pigmented area, NP
16 Presence of hairs on the abdomen
Measure
0-94—1-76
V or
V or
Vor
V or
Vor
Vor
Vor
D V or
V V or
x
x
x
x
x
x
x
x
x
Yolk Yolk ±
Yolk +
Compared with
shade of yolk
as above
V or x
Vor x
15
003-3-5
V or x
each pod were kept until they hatched. The time of hatching was recorded and
the total length of the development period was calculated. This period was taken
as 100 % and the percentage of this period that 24 h represented calculated for
each pod of eggs. In this way the age of each embryo was calculated as a percentage of the total development time of the pod from which it had been removed. All the embryos were then arranged in order of their percentage age,
together with their tables of developmental characters. The fixed material provided a means of direct comparison of each embryo with its neighbours
in the series. Both the fixed animals and the tables of characters formed
an orderly development sequence, despite the fact that they came from six
pods whose incubation times varied from 14-0 days (337 h) to 16-6 days
(400 h). The rate of development in any one pod, therefore, appears not to
fluctuate, even though different pods develop at different rates. The major
Stage of embryonic development
111
changes which occur during development are listed in Table 3. Some of these are
also shown in drawings made from photographs of the fixed material (Figs. 4, 5)
and of eggs in which the shell was made transparent by dissolving the chorion in
3% sodium hypochlorite solution (Fig. 6) (Slifer, 1945).
Fig. 4. Development of the metathoracic leg. The age of the animals from which the
leg was taken is expressed as a percentage calculated from the development time of
the rest of the embryos in the pod.
—
Mean F/bw = 1 -46
Soft tibial spines
Faint (yolk -) spotting
on femur and tips of
tarsi
Femur crescent just
distinguishable
Spotting ± dark as yolk
64-6
67-5
71-2
73-9
75-6
77-8
80-2
80-8
81-9
B
A
B
A
D
B
A
F
E
Spotting darker than
yolk. Tibial spines
more robust. Tips of
tarsi dark brown
Mean F/bw = 1 -25
—
Mean F/bw =1-0
Tibia slightly curved
—
61
Mean F/bw = 0-93
Tibia S-shaped
Leg
A
Calculated
age
Pod
(%)
Spots on thorax ± dark
as yolk
Spots on thorax darker
than yolk
(yolk - )
Scattering of spots on
lateral margin of thorax
Difficult to see yolk
through legs
Yolk visible only through
the dorsolateral body wall
Eye almost fully pigmented. P/NP = 2-85
Dorsal half of eye pigmented. Mean P/NP
= 0-95
Mean P/NP = 2 0
Small dorsal crescent
Yolk easily visible
through both the dorsolateral body wall and legs
Tissues more robust but
yolk still visible as above
Eye
Body
Teeth well formed but
not pigmented
—
Teeth forming along
inside edge
Crinkling of edge of
mandible marked
Definite teeth formed
at tip of mandible
Crinkling of edge of
mandible
Teeth absent
Teeth absent
Teeth absent
Mandible
Head
Spots darker than yolk
Spots ± dark as yolk
—
Scattering of spots
on head (yolk -)
—
Almost normal size
Small relative to body
Table 3. The major morphological changes which occur during the later stages of development
7*
TYRE]
Increase in numbers of
spots and hairs on legs
M e a n F/bw = 1 -76
86-8
86-9
881^
89-4
90-6
90-9
92-8
93-4
96-3
97-5
97-7,
100
F
A
E
D
C
B
F
A
D
B
C
A
Pigmentation of femur
crescent just beginning.
Tibial spines and
tarsal claws pigmented
Pigmentation of femur
crescent almost
complete
M e a n F/bw = 1-7
M e a n F/bw = 1-55
84-4
B
General increase in
pigmentation
82-5\
83-5
Leg
D
C
Calculated
age
Pod
(%)
/
'"
pigmentation
General
increase
Hairs present on the
abdomen
Increase in numbers of
spots and hairs on
thorax and abdomen,
although spots on abdomen few compared
with thorax and legs
Hatching
1
Well denned
spot at
base of
/
cercus
\
Occasional (yolk—) spots
on the abdomen
—
N o longer possible to
discern the margins of
the gut through the
dorsolateral body wall
Body
—
—
—
—
—
—
—
Eye fully pigmented
P/NP = 3-5
—
Eye
Table 3 cont.
Teeth fully pigmented
but slightly redder than /
in hatchling
Tips of teeth well
pigmented
Tips of teeth just
pigmented
Mandible
Prominent hairs on
antennae
Increase in the number
of spots and hairs
General increase in
pigmentation
Head
os
ni
Co
714
N. M. TYRER
•I* v V K) y V ?
61-0%
67-5%
71-2%
77-8%
81 9%
93-4%
100%
Fig. 5. Development of the mandible. The anterior view of the left mandible is shown.
The age of the embryos from which the mandible was taken is expressed as a percentage calculated from the development time of the rest of the embryos in the pod.
1 mm
Fig. 6. Drawings from photographs of eggs staged on morphological criteria. The
shell was made transparent by treating with 3% sodium hypochlorite (Slifer, 1945).
Stage of embryonic
development
715
DISCUSSION
Calculating the percentage development from measurement of the incubation
period enables any point in development to be defined accurately. For some
purposes, however, the method is more elaborate than necessary and the construction of special apparatus may not always be justified. Where precision is not
required it is convenient to distinguish five morphological stages. These are
easily identifiable and have approximately equal intervals of development between them. Drawings of these stages of development are shown in Fig. 6. Work
on embryos staged by either of the two methods can be related since the percentage development of the morphological stages is defined.
The 60% stage
The most important criterion for defining this stage is the small size of the head
and legs in relation to the thorax and abdomen which have swollen to accommodate the yolk. The metathoracic femur is not as long as the body is wide, and
the head is so small that the eyes protrude laterally and dorsally. The eye pigment
is confined to a small orange crescent dorsally and the mandibles are not differentiated into teeth. The tissues are sufficiently transparent for the yolk to be visible
through the legs. The range of development over which embryos display these
characters was not determined accurately as it was found in physiological and
behavioural investigations (Tyrer, 1968) that this was not a particularly important stage.
The 70% stage
The head is almost as large as in the hopper and the metathoracic femur is as
long as, or slightly longer than, the body is wide. The tissue of the legs now makes
it difficult to see the yolk through them, although this is still clearly visible
through the body wall of the abdomen. The eye is two-thirds pigmented but the
mandibles do not yet show differentiation into teeth. The range over which
embryos show this combination of characters is small. The eye is less than half
pigmented in the 67-5% stage and by the 71-2% stage the mandibles show a
marked crinkling along the edge where the teeth will form.
The 80 % stage
The metathoracic femur is now half as long again as the body is wide and
near the joint with the tibia a crescentic mark is just distinguishable on the
cuticle of the femur. Soft unpigmented spines can be seen on the tibia if the
embryonic cuticle is dissected off (see Fig. 4). The tips of the tarsi are just pigmented and pale pigment spots have appeared on the legs. The mandibular
teeth are easily distinguishable. These characters are visible from the 77-8%
stage onwards, but an upper limit can be put on this stage when the pigment
spots on the legs become as dark as, or darker than, the yolk. At this stage also
(81-9%) the tibial spines are easily visible through the embryonic cuticle.
46-2
716
N. M. TYRER
The 92 % stage
The stage between 90-9 and 92-8 % is the most easily defined. Black pigment
begins to appear in the femur crescent at the 91 % stage and this region is fully
pigmented by the 93 % stage. A very dark pigment spot also appears at the base
of the cercus during the same period. The ratio of the metathoracic femur
length to body width is nearly at the hatchling value of 1-76. Pigmentation of the
tibial spines and the tarsal claws is complete and that of the teeth almost complete. The head, thorax and legs are well pigmented and the tissues are opaque,
so the yolk is no longer visible except in the dorsal mid-line where the heart lies.
The intensity of pigmentation of the head, thorax and legs continues to increase
up till hatching, but few pigment spots appear on the abdomen, the colour of
which appears green to the naked eye.
Table 4. Relation between the stages defined by Shulov & Pener (1963) and
those defined in this investigation
Stage XX
Stage XXI
Stage XXII
Stage XXIII
'The embryo occupies the 'The mandibles are almost
always provided with
whole length of the egg.
conspicuous teeth. The
Red pigment is present
hind tibia bears longiin the dorsal part of the
tudinal rows of spines.'
eye. The hind limb is almost straight, longer
than and nearly parallel
to the femur. The distal
spurs of the hind tibia
and the claws of the hind
tarsus are developed.'
The whole eye is deep red.
'The antennae and legs are
The spines of the tibia
gradually becoming
darker than the rest of the are black-brown. Dark
crescents are apparent
body. The teeth of the
on the distal end of the
mandibles and the claws
hind femur.'
of the tarsi are conspicuously darker than
adjacent parts.'
Calculated 67-2±2-8%
Calculated 8 4 ± 2 - l %
Calculated 78-4±2-8%
Calculated 89-6%
standard deviation
not given
The 100% stage
This stage is defined as the period between emergence from the egg and the full
darkening of the cuticle, which normally lasts about 2 h. The light green colour
of the newly emerged hopper darkens within an hour and by 2 h the animal is
almost black.
Using these criteria it was found that the percentage development could be
estimated usually to within 2 % of the development time (Tyrer, 1968), even in
the early stages in which few samples were taken. Definitions of intermediate
stages, however, are best obtained by accurate measurement of the age of the
embryo and calculating this as a percentage of the development period as
described above.
Shulov & Pener's descriptions of their stages for the same period of development described here are given in Table 4 together with the equivalent percentage
age which has been calculated from their data. From their figures the calculated
Stage of embryonic development
111
percentage age of their XX stage is 67-2 % ±2-8 % of the XXI stage 78-4 % ±
2-8 %, of the XXII stage 84 % ± 2-1 % and of the XXIII stage 89-6 % (standard
deviation not given). The morphological criteria for the stages they define,
although imprecise, correspond well with the characteristics defined in this study.
SUMMARY
1. The length of embryonic development in Schistocerca gregaria was measured
using specially constructed recorders to determine the time of oviposition and
the time the first egg in a pod hatched.
2. A variation in development time of 330-410 h was found in eggs kept at
28 °C.
3. To relate embryos from pods with widely differing development times, their
age was expressed as a percentage of the development time of the rest of the eggs
in the pod.
4. The validity of the method was checked by comparing the external features
of embryos removed at 24 h intervals from 6 egg pods with widely differing
development times. When arranged in order of their percentage age the external
features formed an orderly development sequence.
5. From the data obtained in this study, five easily identifiable stages have been
defined in the later part of development in which external changes are slight.
These stages can be used when the more accurate method, which requires exact
measurement of the development period, is not required.
RÉSUMÉ
Estimation quantitative du stade de développement embryonnaire
du Criquet: Schistocerca gregaria
1. La durée du développement embryonnaire de Schistocerca gregaria a été
mesurée en utilisant des enregistreurs construits spécialement pour déterminer
le moment de la ponte et le moment de l'éclosion du premier œuf.
2. On a trouvé, dans des œufs maintenus à 28° une variation de durée de
développement s'étendant de 330-410 h.
3. Pour exprimer les relations entre les embryons des pontes avec des durées
différentes de développement, l'âge de chaque embryon a été défini en pourcentage de durée de développement par rapport à celle du reste de la ponte.
4. La validité de la méthode a été contrôlée en comparant les formes extérieures
d'embryons prélevés à 24 h d'intervalle et provenant de 6 pontes à durées de
développement très différentes. Lorsque ces œufs ont été classés suivant le
pourcentage de durée, les formes extérieures se rangeaient suivant une séquence
ordonnée de développement.
5. D'après les données obtenues par cette étude, cinq stades bien identifiables
ont pu être définis pour la partie avancée du développement, au cours de laquelle
718
N. M. TYRER
les changements externes sont faibles. Ces stades peuvent être utilisés lorsque la
méthode plus exacte, qui requiert la mesure exacte des périodes du développement, n'est pas exigée.
This work was carried out as part of a Ph.D. research project at the Department of Zoology,
at the University of Cambridge and was supported by a grant from the Agricultural Research
Council. I should like to thank my supervisor, Dr J. E. Treherne, for his advice and
encouragement.
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(Manuscript received 18 June 1969)