J. Embryol. exp. Morph. Vol. 54, pp. 47-74, 1979
Printed in Great Britain © Company of Biologists Limited 1979
47
Quantitative staging of embryonic development of
the grasshopper, Schistocerca nitens
By DAVID BENTLEY,1 HAIG KESHISHIAN,
MARTIN SHANKLAND AND ALMA TOROIAN-RAYMOND
From the Department of Zoology, University of California, Berkeley
SUMMARY
During development of the grasshopper embryo, it is feasible to examine the structure,
pharmacology, and physiology of uniquely identified cells. These experiments require a fast,
accurate staging system suitable for live embryos. We present a system comprising (1)
subdivision of embryogenesis into equal periods, (2) expression of stage in percent of complete
embryogenesis time, (3) characterization of stages by light micrographs (and descriptive
text), and (4) illustration of stages at the egg, embryo, and limb levels of resolution. Advantages
of a percent-system include communicability, flexibility in temporal resolution, accurate
assignment of elapsed time in developmental processes, and uniform coverage of the period
of embryogenesis. The stages described are at 5 % intervals with an estimated error of ± 1 %.
INTRODUCTION
Recently it has become possible to investigate the physiology, pharmacology,
and morphology of single, identified neurons, neuroblasts and other cell types
during embryogenesis in grasshoppers (Bate, 197'6 a, b; Spitzer, 1979; Goodman
& Spitzer, 1979; Goodman, O'Shea, McCaman & Spitzer, 1979; Bentley &
Toroian-Raymond, 1979). The paucity of preparations in which these approaches
are feasible has made grasshopper embryogenesis particularly attractive for
analysing many problems in developmental neurobiology and developmental
biology in general. To accurately characterize the time course of developmental
events, it is necessary to have a precise, rapid staging system, applicable to
unstained, living material and covering the entire period of embryogenesis.
Such a staging has not been available.
There is an extensive literature on grasshopper embryogenesis extending
from the mid-nineteenth century (Packard, 1883; Wheeler, 1893; Slifer, 1932a;
Roonwal, 1936; Johannsen & Butt, 1941; Anderson, 1972). Several systems for
staging development have been described (Table 3). Although various features
of these systems are excellent, no single one covers the entire course of embryogenesis with the temporal and cellular detail now required. Most descriptions
1
Author's address: Department of Zoology, University of California, Berkeley, CA 94720,
U.S.A.
48
D. BENTLEY AND OTHERS
are illustrated by camera-lucida or free-hand drawings of whole eggs or embryos;
many cover only a portion of embryogenesis. Most stages have been marked by
easily observable changes in external morphology or orientation of the embryo;
as these events are not distributed uniformly throughout development, relatively
long, uncharacterized periods occur in these systems.
In this paper, we present a new staging system. Its main features are: (1) the
stages evenly subdivide the period of development; (2) stages are expressed as a
percentage of total developmental time; (3) stages are illustrated by light
photomicrographs; (4) stages are illustrated at three levels of increasing resolution (egg; embryo; limb).
MATERIALS AND METHODS
The experimental animals were Schistocerca nitens, initially captured in 1963
and maintained in culture for approximately 50 generations. The animals were
raised in small, crowded cages at 31 ± 1 °C, 16L/8D light cycle, and 60 ± 5 %
relative humidity on a diet of freshly sprouted wheat supplemented by wheatgerm and dry dog-food. Generation time was 10-12 weeks.
Egg-pods were deposited in 6 cm diameter/10 cm high paper cups containing
cleaned no. 1 sand moistened by 15 % water by weight. Cups were capped during
incubation. Pods contained from 25 to 100 eggs. While pods used for maintaining the culture could be left undisturbed, those intended for experiments had
to be opened. Two methods were used for accomplishing this. In the first,
the pod was placed on top of the moist sand, broken into several clusters of
exposed eggs, and covered with damp cotton; alternatively, the pod was completely dispersed and eggs were individually washed in distilled water and placed
separately on filter paper kept at a constant moisture level by capillary wetting.
Eggs were kept at various temperatures in an incubator accurate to ± 0-5 °C,
and at a relative humidity of 60 ± 2 %.
We wanted to express developmental stage as a percentage of total developmental time, and further, to place the stages at equal intervals throughout
embryogenesis. To accomplish this, it was necessary to have a group of synchronously developing eggs whose total developmental time could be accurately
predicted. Sample eggs could then be withdrawn from this group at equal
intervals (5 % of total developmental time in this case) and described.
Eggs within the same pod formed our synchronous groups. The total developmental time of each egg was determined with an accuracy of ± 45 min. Grasshopper eggs are fertilized when deposited (McNabb, 1928; Slifer & King, 1934)
and completion of a pod takes about 1 h. We selected only pods where deposition
was observed, so the time of fertilization could be determined to ± 30 min.
Hatching time was established by continuously observing each pod and counting
and removing all the nymphs which hatched within each 30 min period; the
hatching time of each nymph was consequently known to ±15 min. This
Quantitative staging of embryonic development of Schistocerca 49
information was obtained from ten pods, and formed the data for a quantitative
characterization of the synchrony of hatching within pods (Figs. 1, 2).
Five percent staging required the selection of 20 equally spaced observation
times during the complete period of development. This was done by determining
the temperature at which the eggs developed in 20 days, and then observing the
eggs each day at the time of initial deposition of the pod. The appropriate
temperature was predicted by observing the development of 192 pods at
temperatures ranging from 30 to 35 °C. The actual developmental time of each
of the pods used for staging was determined as described above.
The staging descriptions are designed to be pre-experimental, and therefore
are based on features which can be seen in unstained, living embryos with a
dissecting microscope. Correspondingly, all photomicrographs were made with
a dissecting microscope (Wild M5A; a few additional features which can be
seen in simple squashes in a compound microscope are noted). A text description
of each stage is provided as well as light micrographs at three levels of resolution:
(1) the whole egg showing the size, location, and orientation of the embryo;
(2) the embryo; (3) detail of the metathoracic leg. Eggs were immersed in 3 %
sodium hypochlorite for 1 min; this procedure clears the chorion but doesn't
remove it (longer exposure clears better but causes osmotic changes which alter
the shape of the embryo). Cleared eggs were photographed in dark-field
illumination; embryos were photographed in transmitted illumination or, after
they became opaque, in incident illumination. All colors are described from
incident illumination.
The saline in which embryos were examined comprised NaCl 140 mM,
KC1 5mM, CaCl 2 .2H 2 O 4mM, MgSO 4 .7H 2 O 2mM, TES 2 HIM, dextrose
55-65 mM, pH 7-2, osmolarity; 310-325 milliosmol/kg. High osmolarity was
crucial to maintaining physiological condition and had to be adjusted for age of
the embryo (Carlson, 1961; Grellet, 1968). It was altered by varying dextrose
concentration to maximize heart rate, peristalsis rate, neuroblast mitosis
frequency or, in early stages, to prevent shrinking or swelling of the amniotic
cavity.
The final staging is based upon precisely timed observation of eggs from
eight pods. At each observation period, descriptions of several eggs from each
pod were made; for three pods, photographs of several eggs were made each
day. Confirmatory observations have been made on many additional pods.
RESULTS
The synchrony of hatching of eggs within the same pod is shown in Fig. 1.
These four pods were maintained on sand under moist cotton (first method);
the cotton was removed a few hours before the onset of hatching. The degree of
synchrony can be expressed by calculating the percentage of eggs which hatched
within a period equal to 1 % of total developmental time (Fig. 1). In three of four
50
D. BENTLEY AND OTHERS
10
Pod C
n = 50
7o= 100
Pod I)
'.'o= 100
•3 40
30
20
10
-
19
20 19
Developmental time (days)
2G
Fig. 1. Hatching synchrony of eggs within each of four pods (A, B, C, D; incubated
on sand), n = the number of eggs hatching from each pod; % = the percentage of
hatching eggs from each pod which hatched within a period equal to 1 % of the
total development time.
Quantitative staging of embryonic development of Schistocerca 51
20-5
Development time (days)
Fig. 2. Hatching synchrony of eggs within each of four pods (E, F, G, H; incubated
on filter paper), n = the number of eggs hatching from each pod; % = the percentage of hatching eggs from each pod which hatched within a period equal to 1 %
of the total developmental time. In pod H, seven eggs hatched when all the eggs were
wetted with cool water at day 19; all of the remaining eggs hatched within the 9 h
period shown, but the exact time of hatching was not noted.
pods, all the eggs hatched within this period. This degree of synchrony indicates
that the time of hatching of a subset of eggs from a pod is an accurate estimate
of when eggs removed for staging descriptions would have hatched if they had
been left undisturbed. Therefore, the percentage of developmental time
experienced by embryos withdrawn for staging can be estimated within an error
ofl%.
An additional problem is the degree to which synchronous hatching indicates
synchronous development. Mechanisms, such as pheromonal or mechanical
stimulation, might be present which initiate simultaneous hatching in eggs that
are actually at slightly different stages of development. To evaluate this factor,
52
D. BENTLEY AND OTHERS
Fig. 3. Developmental synchrony between eggs from the same pod at different stages
in embryogenesis. Metathoracic limbs of four embryos from the same pod are
shown at 55 % development and at 90 % development. The degree of similarity
between these limbs is representative of the range of variation normally encountered
and indicates that synchrony falls well within the 5 % range throughout embryogenesis. Transmitted illumination.
Quantitative staging of embryonic development of Schistocerca 53
Table 1. Features of 5 % developmental stages
Stage
Characteristics (distinguishing from previous stage)*
0%
5%
10 %
15 %
20 %
Egg light yellow; lipid droplets uniform in size
Egg brown; lipid droplets variable in size; energids in posterior yolk
Disc-shaped blastoderm; energids throughout yolk
Embryo cephalized; amnion present; early yolk cleavage
Primary segmentation; embryo post-anatrepsis; yolk cleavage about two thirds
length of egg
Segmentation to third abdominal segment; neuroblasts visible; leg rudiments
larger than mouthpart rudiments
Embryo fully segmented; eye region delaminated; rudiments onfirsttwo
abdominal segments
Rudiments on all abdominal segments; primary cuticle visible; proctodeum in
tenth abdominal segment
Separation offemur and tibia in metathorax; embryo half length of egg;
proctodeum in ninth abdominal segment
Separation of tibia and tarsus in metathorax; katatrepsis initiated; proctodeum
in eighth abdominal segment
Pigmentation of eye-plate; embryo post-katatrepsis; metathoracic tibia parallel
to femur
Antennal furrows; metathoracic tibia reaches base of femur; herringbone array
of extensor tibia muscle fibers
White line on eye-plate; rotation of embryo; embryo more than three quarters
length of egg
Double curvature of metathoracic tibia; leg twitching; synchronous contraction
of median sinus
Metathoracic tibia straightened; embryo pale green; white line close to anterior
margin of eye
Longitudinal rows of brown pigment spots line metathoracic femur; brown
pigment on dorsal, caudal midline
Brown pigment spots on all legs; apolysis of second embryonic cuticle completed
Clearing offemoral crescent; embryo bright green; vertical eye stripes
Tarsal claws are black; brown pigmentation covers compound eye; transverse,
dark-green stripes on metathoracic femur
Black hairs on antennae; integument opaque white; dark blue color within
antennae and metathoracic legs
Egg hatches; in saline, embryos near 100 % begin peristaltic contractions
* Italics indicate most unequivocal feature.
25 %
30 %
35 %
40 %
45 %
50 %
55 %
60 %
65 %
70 %
75 %
80 %
85 %
90 %
95 %
100 %
we examined groups of embryos from the same pod at various times in development. Inspection of rapidly changing features, such as the degree of differentiation of the metathoracic limb (Fig. 3), indicated that most embryos within a pod
were at a very similar stage of development. Occasional out-of-step embryos
were encountered. These were almost invariably behind their pod-mates,
suggesting that they may have been moribund individuals which would have
failed to hatch. Significant variability in the numbers of such individuals
occurred between pods.
We observed pods which hatched in total developmental times ranging from
about 19-5 to 20-5 days. This gave a good sampling of the developmental rates
54
D. BENTLEY AND OTHERS
surrounding and including 20 days. Daily sampling at these rates provided the
basis for our characterization of 5 % developmental stages. The photomicrographs (Figs. 3-9) were made from pods characterized in Fig. 2. Key features
delineating each 5 % stage are briefly summarized in Table 1. The following
is a more detailed description of each stage:
0%
Freshly deposited eggs are light yellow. Within about 3 h, they tan to a dark
brown. Eggs on the exterior of the pod tan first. The change in coloring is due
to a darkening of the outer egg envelope or chorion, which is initially transparent
and reveals the yellow, yolk-filled interior of the egg.
Yellow, lipid yolk droplets (Mahowald, 1972) are initially uniform in size,
about 20 ± 5 /mi in diameter (be aware that they will begin to fuse when expressed from the egg into saline). As the egg develops they become much more
heterogeneous.
The poles and axes of the egg are assigned by convention with respect to
the orientation of the egg in the maternal ovariole (Mahowald, 1972; Anderson,
1972). The posterior pole is marked by a prominent, opaque, cap-like etching
of the chorion (Fig. 4-5). At the base of this cap are the micropyles through
which the sperm enter the egg. The anterior pole lies at the opposite, more
pointed, end of the egg. Eggs are usually curved. The concave side is dorsal
and the convex side ventral (Fig. 4 - 5 0 ) ; however, uncurved or doubly curved
eggs are regularly found.
5%
The egg is brown. Lipid yolk droplets are highly variable in diameter, ranging
from 10 to 100 /«n (Fig. 6 - 5).
Dispersed among these droplets in the region of the posterior pole are
colorless, oblong islands of cytoplasm, often containing round nuclei about
40 jam in diameter. These are the cleavage energids from which the embryo,
embryonic membranes, and yolk cells will develop (Anderson, 1972). Energids
cannot be detected through the cleared chorion; they are most easily seen by
puncturing the appropriate part of the egg, squashing the expressed yolk under
a coverslip, and viewing in a compound microscope.
10%
At the posterior pole, located toward the ventral side of the egg, is a small
cluster of cells floating on the surface of the yolk droplets (Fig. 6 - 10). The
cluster is a disc-shaped monolayer about 300 /im in diameter and comprises the
primitive blastoderm, from which the embryo will develop.
Cleavage energids are now distributed throughout the entire length of the egg,
although they are more numerous toward the posterior pole.
Quantitative staging of embryonic development of Schistocerca 55
From the outside of a cleared egg, the embryo is now visible on the
posterior/ventral surface (Fig. 4 - 1 5 ) . Its dorsal side is apposed to the yolk and
the anterior end faces the anterior pole of the egg.
The embryo has differentiated a widened, anterior, cephalic region (protocephalon) extending about half its length (Fig. 6-15). Antennal and preantennal segments will arise here. Mouthpart, thoracic, and abdominal segments
will develop from the narrow, posterior end of the embryo (protocorm).
The embryo is one cell layer thick except for a band of cuboidal cells on the
dorsal midline forming a second, inner layer. This inner layer extends about
two thirds the length of the embryo, from the posterior end to the mid-protocephalon. At its anterior end, it expands to form two lobes. The inner layer
arises from invagination of cells during gastrulation (Roonwal, 1936; Anderson,
1972) and comprises the presumptive mesoderm.
A thin membrane (the amnion) is attached to a ridge of columnar cells at
the perimeter of the embryo (Fig. 6 - 1 5 ) and covers the entire ventral surface.
This membrane and the extra-embryonic membrane (the serosa) arise at a
slightly earlier stage by the fusion of amniotic folds (Roonwal, 1936). The serosa
eventually forms a sac enclosing the yolk (50 %; Fig. 7 - 55).
The yolk remains a dispersion of variably sized droplets except at the extreme
posterior pole where large, oblong cells, from 200 to 300 /*m in length, mark the
onset of yolk cleavage. The yolk is gradually ingested by the formation of these
cells in a posterior-anterior progression.
20%
The location of the embryo has shifted due to an immersion and rotation
within the yolk (anatrepsis; Wheeler, 1893). The embryo is now on the dorsal
side of the egg, with its anterior end facing the posterior pole and its dorsal
surface apposed to the yolk.
The protocorm has elongated and differentiated into two distinct regions
separated by a slight flexure and thickening of the embryo (Fig. 6 - 20). The
anterior region contains the presumptive mouthpart tissue, while the posterior
region will give rise to the thorax and abdomen.
The protocephalon has a medially located depression on its ventral surface,
the oral opening (stomodeum). A pair of small protruberances slightly posterior
and lateral to the stomodeum mark the antennal rudiments.
The cleavage of the yolk has progressed to approximately two thirds of the
length of the egg, giving the yolk the appearance of a cellular mosaic (Fig. 4 - 20).
Anterior to this area, the yolk still consists of variably sized droplets.
56
D. BENTLEY AND OTHERS
25%
Mouthpart, thoracic, and the first three abdominal segments are now clearly
delineated (Fig. 6 - 25). The segments in the mouthpart and thoracic regions
each possess a pair of ventrally placed protruberances, the limb buds.
The thoracic limb buds as a group are larger than the mouthpart limb buds,
with the largest pair formed by the metathoracic segment. The metathoracic
limb bud consists of an inner mass of cuboidal cells surrounded by a rind of
tightly packed columnar cells (Fig. 8 - 2 5 ) ; all limb buds appear this way
when they first differentiate. Anterior to the stomodeum is a small protruberance, the presumptive labrum. The antennal rudiments, lateral to the stomodeum, have enlarged and turned medially. The abdominal segments at this stage
lack limb buds.
The mesoderm has become divided into a series of hollow, segmental blocks
of tissue in both the mouthpart and thoracic regions. Mesodermal tissue in the
abdomen is flattened.
The unsegmented caudal tip of the embryo possesses a small invagination,
the primitive anus (proctodeum).
Along the ventral, median surface is a band of large spherical cells, neuroblasts (Wheeler, 1893; Carlson, 1961; Bate, 19766). In the protocephalon they
are distributed in packets anterior and lateral to the stomodeum, while in the
protocorm they are arranged in a metamerically repeated pattern.
Cleavage of the yolk has been completed.
30%
The embryo is fully segmented (Fig. 6 - 30). The first two of the eleven
abdominal segments possess limb buds (note that the lateral edge of the segment
is easily mistaken for the limb bud, which lies more medially on the ventral
surface). The metathoracic leg has elongated and has a slight, medially directed
bend (Fig. 8 - 30). The mesothoracic and prothoracic legs are about the same
size, but smaller than the metathoracic leg. The labial and maxillary rudiments
are also of equal size, and are about twice as long as the mandibular rudiment.
A thin membrane, the provisional dorsal closure, extends across the dorsal
surface of the embryo. This membrane is supplanted later by a true, dorsal
ectoderm. On the ventral side of the embryo, the amnion is stretched tightly
over the segmental appendages, folding them medially.
The head capsule has two prominent lobes which will eventually be occupied
by the compound eyes. The posterior portion of these lobes has delaminated
into two layers separated by a space. The outer layer is the eye plate (Roonwal,
1937) which comprises the presumptive retina and associated ommatidial
structures; the inner layer will become the distal portion of the optic lobe of the
brain. The eye plate is curved, and its posterior (medial) margin is thickened.
Quantitative staging of embryonic development cf Schistocerca 57
35%
Limb buds occur on all eleven abdominal segments. Those of the first
segment, the pleuropodia, are multi-lobed and are much larger than buds of
other segments. They are embryonic structures lost at hatching. Limb buds
of segments 3-10 are ventral, while the remaining buds (1,2,11) extend laterally.
The primary cuticle covers the surface of the embryo, and is visible as a clear
film stretched over the appendages and into the opening of the proctodeum.
This cuticle is later detached from the epidermis (apolysed) and replaced by a
secondary cuticle which is shed after hatching (Mueller, 1963; Micciarelli &
Sbrenna, 1972).
A slight invagination of the columnar cell rind is visible at the tip of the
metathoracic leg. It marks the beginning of differentiation of the claw retractor
tendon (apodeme), one of three leg tendons (Snodgrass, 1929). Maxillary and
labial limb buds have become trilobed.
A pair of neurons, the pioneer fibers (Bate, 1976 a), can be seen within each
antenna. The cell bodies are located at the tip of the antenna, adjacent to the
columnar cell rind. Similar cells arise in other appendages.
In each segment, neuroblasts have proliferated clusters (presumptive ganglia)
of ganglion mother cells and undifferentiated neurons (Bate, 1916 b; Goodman
& Spitzer, 1979). Fine, intersegmental fibers run longitudinally on both sides
of the midline in segments anterior to the abdomen. They are viewed most
easily from the dorsal aspect, and comprise early, axonal outgrowths of ventral
cord neurons.
The proctodeum has invaginated to the anterior border of the tenth abdominal
segment (Fig. 6 - 35).
40%
The embryo is visible as a wedge extending from the posterior pole for about
half the length of the egg (Fig. 4 - 40). The metathoracic leg has a notch on its
medial side (this will be the ventral side of the adult leg) which separates the
femoral and tibial regions (Fig. 8 - 40). The claw retractor tendon has extended
further proximally from its invagination, and the leg is heavily invested with
spindle-shaped cells (probably myoblasts) and nerve fibers. These features also
occur in the other thoracic and mouthpart appendages.
In the central nervous system, intersegmental fiber bundles extend along the
entire length of the embryo. Transverse, intrasegmental nerve fibers can be seen
in all segments.
The proctodeum has invaginated to the anterior border of the ninth abdominal
segment (Fig. 6-40).
58
D. BENTLEY AND OTHERS
45%
The embryo has begun moving around the posterior pole of the egg
(Fig. 4 - 45). At the completion of this movement (katatrepsis; Wheeler, 1893;
Slifer, 19326; Anderson, 1972), it will face the anterior pole of the egg and its
ventral surface will appose the ventral side of the egg.
During katatrepsis, the embryo generates a series of rhythmical, metachronal
waves of posterior to anterior contractions. The waves can be seen in cleared
eggs, and occur at 10-20 times per minute in 25 °C saline. They continue in less
pronounced form after the completion of katatrepsis.
The metathoracic leg has an additional constriction separating the tibial and
tarsal regions (Fig. 8 - 45). Two tendon invaginations are just noticeable on the
femur, the extensor tibia tendon on the lateral aspect and the flexor tibia
tendon on the medial (Fig. 8 - 45, 50). The antennae show the onset of segmentation, with four thickenings of the outer, columnar rind. The eleventh
abdominal appendages, cerci, have broadened into flattened lobes on either side
of the proctodeum (Fig. 6 - 45).
The posterior margin of the eye plate contains spindle-shaped cells extending
across its full depth. These appear to be retinula cells of the differentiating
ommatidia. Large number of fibers cross to the distal surface of the optic lobes.
Clusters of white cells mark the appearance of the lateral ocelli.
The fibers of the ventral nerve cord have formed a ladder-like arrangement,
with a pair of distinct bundles of transverse fibers in each segment intersecting
the longitudinal, intersegmental bundles on both sides of the midline.
On either side of the midline, the dorsal surface of the embryo is flecked with
white spots. These spots are composed of heavily pigmented cells in a tissue
layer which becomes the fat body.
The proctodeum has invaginated to the anterior border of the eighth
abdominal segment (Fig. 6 - 45).
50%
The embryo has completed its movement around the posterior pole
(Fig. 4 - 50). Anterior and dorsal to the head is a plug of yolk enclosed by the
serosa and occupying about half the volume of the egg.
The metathoracic leg has flexed so that the tibia is parallel to the femur
(Fig. 8 - 50). Spurs are visible at the distal end of the tibia. The tarsus has
divided into two segments (this division has not yet occurred in the other legs).
The cerci have enlarged and formed a nipple-like process at the tip. Sexual
differences in the genitalia can be distinguished (Karandikar, 1942).
The eye plate has an unlayered red-brown pigment along its posterior margin
(eye axes will be given with respect to adult eye position). Differentiation of
retinula cells has proceeded about half way to the anterior edge of the eye.
The median ocellus is present. In the ventral nerve cord, a broadened, fibrous
Quantitative staging of embryonic development of Schistocerca 59
region occurs at the intersection of the fiber bundles. This region is the incipient
neuropil of the embryonic ganglia.
There is marked apolysis of the primary embryonic cuticle, with the secondary
cuticle forming underneath (Mueller, 1963; Micciarelli & Sbrenna, 1972).
The proctodeum has invaginated to the anterior border of the seventh
abdominal segment.
55%
The ventral aspect of the embryo is still adjacent to the ventral (convex)
side of the egg. The head extends between half and two thirds of the distance
to the anterior pole (Fig. 5 - 55). The amnion now closes the dorsal surface
up to the prothorax (Fig. 7 - 55). Yolk is continuous from the serosal sac
through the open cervical dorsum into the midgut of the embryo. The yellow
midgut yolk is encased by transparent fat-body tissue containing a profusion of
white cells (Fig. 7 - 55, 60).
A clear band along the dorsal midline of the embryo demarcates the median
blood sinus, antecedent to the heart. Peristaltic, anteriorly directed constrictions
of this sinus are a continuation of the rhythmical activity first seen at the 45 %
stage.
The metathoracic tibia reaches to the base of the femur (Fig. 8 - 55). The tip
of the leg has a longitudinal furrow marking the tarsal claws. Well-differentiated
muscle tissue is visible in transmitted light. A distinctive, herringbone array of
muscle fibers, the incipient extensor tibia muscle (135a and b; Snodgrass, 1929;
Fig. 8 - 55), occurs along the dorsal (previously medial; 45 %) tendon of the
metathoracic femur. Localized muscle fiber contractions can be seen through
the cuticle, but the leg does not twitch noticeably.
The genital appendages (on the eighth and ninth abdominal segments of the
female and the ninth and tenth of the male) shift toward the ventral midline
(stage 3; Karandikar, 1942). No other abdominal limb rudiments remain between
the pleuropodia and the cerci. Pronounced furrows in the epidermis divide the
antennae into annular segments (Fig. 7 - 55).
A brick-red band starts at the posterior margin of the compound eye and
extends forward one fourth of its width (Fig. 7 - 55). This band comprises both a
superficial and a deep layer of the same pigment. Rows of facets line the surface
of the eye. The superficial pigment accumulates at the borders of the facets,
lending a speckled appearance to its layer. The deeper pigment layer is unbroken,
but bears a pattern of ommatidial silhouettes on its outer surface (in the adult
ommatidium, there are red-brown pigments present in both the photoreceptors
and in two layers of pigment cells that line the outside of each ommatidial
cartridge; Roonwal, 1947).
EMB 54
60
D. BENTLEY AND OTHERS
60%
Usually the embryo has expanded to fill the entire egg except for a small
space at the anterior pole (Fig. 5 - 60). There is considerable variation both in
the completion of expansion and in the amount of space left; about a third of
our embryos did not finish this process until the next stage.
Expansion is accompanied by a 180° rotation of the embryo about its longitudinal axis (Slifer, 19326; Bodenheimer & Shulov, 1951; Jones, 1956), leaving
its ventral surface adjacent to the dorsal (concave) side of the egg (Fig. 5 - 60).
This orientation is maintained for the remainder of embryogenesis. Rotation
of the embryo was confirmed by marking the surface of the egg with wax.
As the embryo fills the egg, the yolk is engulfed by the expanding midgut
(Fig. 7 - 60). The regressing yolk sac transforms into a tubular protruberance
of degenerating serosal cells (dorsal organ; Wheeler, 1893) which sinks into
the midgut to allow the completion of dorsal closure. After closure, a pair
of bilateral bladders, the cervical ampullae, lie between the head and the deeply
wrinkled pronotum. They function during hatching and do not persist into the
first instar (Bernays, 1971).
Genital rudiments of the ninth abdominal segment have partially fused along
the ventral midline in both sexes. A pair of narrow, flattened rudiments are
barely distinguishable at the posterior margin of the female's eighth abdominal
segment, and the tenth segment rudiments of the male have completely disappeared underneath the fused ninth pair. The cercus has elongated into a cone
which lies folded beneath the abdomen (Edwards & Chen, 1979).
A white line extends dorso-ventrally along the anterior margin of the eye
plate (Fig. 7 - 65) which is about halfway across the presumptive compound
eye. An unpigmented zone divides this white line from the parallel posterior
band of red pigment. White pigment cells in each ocellus begin to coalesce
into a disc fronted by a lens primordium.
65%
A narrow space persists between the embryo's head and the anterior pole of
the egg (Fig. 5-65).
The metathoracic tibia assumes a double curvature unique to this stage
(Fig. 9 - 65). The proximal part of the tibia bends away from the femur, and
the distal half gradually curves back toward it. This morphological characteristic is more reliable than behavioral features for identification of the stage
(note: transitional forms obviously occur in the appearance and disappearance
of this and other features; therefore it is important to rely also upon more
subtle changes in the balance of form, such as the relative size of the abdomen
and metathoracic leg, which can be apprehended by studying photographs of
the different stages).
The legs begin to twitch. These periodic jerks are easily distinguished from
Quantitative staging of embryonic development of Schistocerca
61
the smooth limb displacement accompanying bodywall peristalsis. Individual
limbs extend rhythmically at one or more joints, but there is no apparent coordination of frequency or phase between limbs. Limb movements are not
evident until the embryo is removed from the egg.
Peristaltic waves are replaced by simultaneous contraction along the entire
length of the median sinus (Nelson, 1931). Apparently these contractions
represent the beginning of normal heartbeat (Roonwal, 1937, described the
formation of a definitive heart wall within this sinus at an equivalent stage in
Locustd). White fat cells begin to appear beneath the transparent heart tube,
reflecting its detachment from the midgut and the subsequent intrusion of the
fat body.
The labia have shifted medially and fused at the base. Other mouthparts
remain laterally oriented and do not close over the stomodeum until hatching.
70%
Prior to this stage, the embryonic tissue has been transparent or translucent.
The embryo now becomes a very pale green, particularly the head and legs.
The metathoracic tibia retains little of its prominent curvature from the
previous stage (Fig. 9 - 70). The tibia will not become perfectly straight until
after hatching.
Brick-red pigment covers the posterior half of the compound eye (Fig. 5 - 70,
7 - 70). The superficial pigment layer extends further anterior than the deep
layer, forming a speckled, light-red band along its leading edge. A colorless zone
still separates the red region from the anterior white band (Fig. 7 - 70). This
band has also moved anteriorly and approaches the frontal margin of the eye.
In some individuals, brown pigment fringes the cuticular sleeve enveloping
the mandible rudiment (this feature should not be mistaken for the extensive
pigmentation of the mandible teeth that occurs at 90 % development).
75%
This stage is distinguishable by the appearance of longitudinal rows of
brown spots on the metathoracic femur (Fig. 7 - 7 5 ; 9 - 85). The rows contain
about ten spots each, and mark incipient ridges along the dorsal and ventral
edges of the femur. There are no spots yet on the tibia or on the other legs.
Faint brown pigment also appears on the midline of the dorsal body plates
(tergites) of the caudal-most segments.
The anterior white line has reached the frontal margin of the compound eye
(Fig. 7 - 75), and will remain throughout embryogenesis into the first instar.
The red pigment region has turned dark brown and continued to move anteriorly.
The facets are finely outlined in white, and this gives the eye a frosted appearance
under incident illumination.
White teeth begin to form on the medial edge of the mandible rudiment,
within the cuticular sleeve.
5-2
62
D. BENTLEY AND OTHERS
80%
For most embryos, the head is tightly pressed into the anterior end of the
egg (Fig. 5 - 80).
Brown pigment spots are present on the femur and tibia of every leg, but not
on the head or thoracic body-wall. A faint, brown dorsal midline extends
forward to the mesothorax.
The second embryonic cuticle separates from the underlying epidermis over
the entire body surface, and the third cuticle begins to form. The second cuticle
remains intact throughout embryogenesis and is shed soon after hatching
(Bernays, 1972). The third cuticle will be the cuticle of the first instar. Apolysis
of the second cuticle has been described from histological sections for S. gregaria
resembling our stages 70-75 (Micciarelli & Sbrenna, 1972), and can also be
seen in S. nitens embryos of this stage with a compound microscope.
The brown pigment band of the compound eye may encroach upon the
posterior edge of the dorsal spot, a smooth, indistinctly faceted ellipse the size of
an ocellus situated at the dorsal margin of the eye.
85%
The entire embryo is bright green in color, although the midgut yolk still
lends a yellow cast to the abdomen. Brown spots appear on the frontal head,
pronotum, and posterior margins of the meso- and metathoracic tergites.
The dorsal midline darkens.
A large, transparent crescent develops at the distal end of the metathoracic
femur (Fig. 7 - 8 5 ; 9-85), adjacent to the tibial articulation (Tyrer, 1970).
This structure may sometimes be recognized at 80 % development as a small,
indistinct outline that has not cleared.
The brown eye band continues to expand toward the anterior, and now fills
the posterior half of the dorsal spot. The brown color remains dark within the
boundary of this structure, but pales elsewhere except for two vertical stripes
which persist within the light-brown region (Fig. 7 - 85, 90). These stripes will
disappear later and are not antecedent to the postembryonic striations described
by Roonwal (1947). A circular, black image known as the pseudopupil appears
to lie beneath the eye surface (Fig. 7 - 85, 90). The pseudopupil is not a structure
but an optical illusion manifest in the organization of the eye (Horridge, 1977).
Cereal sensory hairs have grown into the empty tip of the cuticular sheath
in some individuals (they are not pigmented and must be viewed in transmitted
light). The presence of sensilla demonstrates the deposition of a definitive
first instar cuticle on the surface of the epidermis (Edwards & Chen, 1979).
90%
Three broad transverse stripes of dark green appear on the metathoracic
femur (Figs. 7 - 9 0 ; 9-90), accentuating the herringbone pattern on the
Quantitative staging of embryonic development of Schistocerca
63
external surface. The pattern reflects a double oblique array of low ridges
overlying the insertions of the extensor tibia muscle fibers on to the inner side of
the cuticle. Brief cuticle indentations occur sporadically within the insertion
area. They are reminiscent of the fiber twitches seen at stage 55, and imply
that functional muscle insertions have been made on to the cuticle (Sharan,
1958). The indentations need not be correlated with whole leg movements.
Light brown pigmentation covers the full width of the compound eye and is
contiguous with the white anterior border. The entire dorsal spot is dark brown.
The tarsal claws and tibial spurs turn black (Fig. 9 - 90), and the cereal hairs
now appear black under incident illumination. The teeth of the mandible, and
sometimes those of maxilla, are a lustrous brown (Fig. 7 - 90). Brown spotting
has progressed to the antennae, tarsi, palps, abdominal tergites, and occipital
area of the head.
95%
The embryonic integument turns opaque white on much of the head and
limbs and on small patches of the body. Brown and black markings are
prominent against this background. The degree of white coloration on the
abdomen varies greatly between individuals, so that the heart may be either
exposed or obscured.
The teeth of both the mandible (Fig. 7 - 95) and maxilla darken to black at
the tips, as do the tibial spines which are pressed flat against the legs by the
embryonic cuticle (Fig. 9 - 95). Black hairs appear on the legs, antennae,
maxillary palps, head, and tergites.
Dark blue tissue appears within the metathoracic legs and the antennae.
This pigmentation is partly concealed by the white color, but the blue tint
is pronounced around the femoro-tibial articulation, beneath the marginal
ridges of the femur, and in the proximal segments of the antennae.
Eye color progressively pales. The vertical stripes have faded, and the eye is
an essentially uniform shade of brown except for the dark dorsal spot.
In most individuals, the spontaneous cuticle indentations seen in the previous
stage are no longer apparent.
100%
No external morphological features unequivocally distinguish this stage from
the previous. The most distinctive difference between the two is their respective
competence to hatch. Hatching is effected by a series of powerful, anteriorly
directed peristaltic contractions of the body wall which rupture the egg and
liberate the embryo still encased in the second embryonic cuticle (vermiform
larva; Bernays, 1971). Hatching competence was quantified by testing embryos
at various stages between 90 % and 100 % from three pods (percentage development was calculated from the median length of embryogenesis for the remainder
of each pod). Embryos were released from the egg under 24+1 °C saline; if
64
D. BENTLEY AND OTHERS
Table 2. Hatching competence of maturing embryos
95 % stage
90 % stage
90-92-5
92-5-95
95-97-5
100 % stage
97-5-100
20
12
15
16
2
0
2
80%
17%
0%
13%
* Showing rhythmical sequences of peristaltic waves (see text).
Number tested
Number hatching"
Percent hatching
Fig. 4. Appearance of live eggs at 5 % developmental intervals during the first half of
embryogenesis (eggs partially cleared in sodium hypochlorite). 5-25, ventral aspect;
30-35, dorsal aspect; 40-50, lateral aspect; posterior pole to right. Arrows: 15,
embryonic disc; 30-40, posterior margin of embryo; 45, embryo turning within
egg; 50, compound eye. Dark-field illumination.
Quantitative staging of embryonic development of Schistocerca
65
Fig. 5. Appearance of live eggs at 5 % developmental intervals during the second
half of embryogenesis (eggs partially cleared in sodium hypochlorite). 55-60, note
that the convex side of the egg has changed from ventral (55) to dorsal (60); this
reflects the 180° rotation of the embryo within the egg. Dark-field illumination.
contractions did not appear, they could sometimes be elicited by brushing the
legs with forceps. Only embryos with the 95 % - 100 % morphology produced
rhythmical sequences of peristaltic waves (Table 2), and the behavior occurred
much more reliably in embryos near 100 %.
Newly hatched larvae normally must dig upward to reach the surface of the
ground (Bernays, 1971). Larvae hatching on the surface begin molting the
embryonic cuticle (first ecdysis) within 10 min. In the first ecdysis, the cuticular
envelope is split along the dorsal midline and the first instar nymph emerges.
Emergence is produced by a complex sequence of actions very similar to that
described for S. gregaria (Bernays, 1972). As the embryonic cuticle is sloughed,
numerous black and unpigmented hairs spring erect, the cervical ampullae
deflate, and the mouthparts close medially for the first time. Following ecdysis,
66
D. BENTLEY AND OTHERS
Fig. 6. Appearance of live embryos at 5 % developmental intervals during the first
half of embryogenesis. 5, site in yolk droplets where embryonic disc will appear;
10 (arrows), embryonic disc. 35, note amnion (membrane) spread out around the
anterior end of the embryo. Transmitted illumination.
the nymph is quiescent for ten to fifteen minutes before taking its initial
steps.
The first instar nymph is bright green with the same brown and black markings
as the embryo. Blue pigment persists within the antennae, but fades from the
metathoracic femur at the time of hatching. Faint vestiges of the embryonic
eye stripes may be visible even after the broad, dark first instar stripe begins to
grow down from the dorsal spot (Roonwal, 1947).
Quantitative staging of embryonic development of Schistocerca
67
Fig. 7. Appearance of live embryos at 5 % developmental intervals during the last
half of embryogenesis. 55, note yolk plug extending anterior and dorsal to embryo
(transmitted illumination). 60-100, incident illumination.
DISCUSSION
Three types of systems have been used previously for staging grasshopper
embryogenesis. Stage has been based upon (1) absolute age of the embryo
(age-staging), (2) distinctive changes in external morphology (event-staging),
or (3) percentage of the total developmental time through which the embryo has
passed (percent-staging). For present purposes, age-staging is unsuitable
because of its inflexibility; it cannot be used when the same species is incubated
at different temperatures, or for different species, or for species with individual
variability in developmental rate. Although the event-staging system may be
more useful for comparing similar stages among widely differing species, we
68
D. BENTLEY A N D OTHERS
Fig. 8. Appearance of a metathoracic limb of live embryos at 5 % developmental
intervals from 25 % to 60 % of embryogenesis. Note that there are marked differences
between each stage, particularly involving segmentation, flexion, invagination
of apodemes, and differentiation of musculature. Transmitted illumination.
Quantitative staging of embryonic development of Schistocerca
69
Fig. 9. Appearance of a metathoracic limb of live embryos at 5 % developmental
intervals from 65 % to 100 % of embryogenesis (65-80, transmitted illumination;
85-100, incident illumination).
prefer the percentage system for studies of developmental processes for the
following reasons: (1) the meaning of a percent-stage is readily apprehended by
non-specialists. This greatly facilitates communication with developmental
biologists who are not entomologists (in practice, the event-staging systems have
been essentially species specific). (2) percent-staging allows greater flexibility in
temporal resolution. If more temporal detail is required, it is straightforward to
extend a 5 % level to a 1 % level of resolution. This kind of modification is a
continuing problem with event-staging systems once the stages have been
initially erected. (3) percent-staging allows an even distribution of stages (at
whatever level of resolution is required) through the entire developmental
70
D. BENTLEY AND OTHERS
Table 3. Stagings of grasshopper embryogenesis
Genus
Schistocerca
Melanoplus
Locusta
Chortoicetes
Ornithacris
Pyrgomorpha
Nomadacris
Locustana
Euprepocnemis
Dociostaurus
Austroicetes
Aulocara
Acrida
Reference
Jhingran (1947), Shulov & Pener (1963), Micciarelli-Sbrenna (1969),
Tyrer (1970)
Nelson (1931, 1934), Slifer (1932o), Salt (1949), Riegert (1961)
Roonwal (1936, 1937), Shulov & Pener (1959), Salzen (1960)
Wardhaugh (1978)
Chapman & Whitham (1968)
Chapman & Whitham (1968)
Shulov (1970)
Matthee (1951)
Khalifa (1957)
Bodenheimer & Shulov (1951)
Steele (1941)
Van Horn (1967)
Kucukeksi (1964)
period. The temporal placement of event-stages is dictated by the occurrence of
easily recognized events and always results in a non-uniform distribution of
stages. Since cellular and sub-cellular processes of great importance in differentiation may not be coupled to these easily recognized events, it is better to
have a staging system which does not leave gaps in the complete course of
embryogenesis. (4) percent-staging permits accurate assignment of elapsed time in
developmental processes. Using event-staging, it is possible to order observations but not to discuss the real or relative durations of processes; it is meaningless to consider elapsed time between two observations unless both are made
in the same animal. A percent system makes it possible to construct the developmental history of a process, using data collected from many individuals, with
recognition of the time between succeeding steps.
The percent-staging system presented here can be correlated with previously
described systems (Table 3). Tyrer (1969, 1970) employed a percent-staging
system at a 10 % level of resolution for the last 40 % of embryogenesis of
S. gregaria. Shulov & Pener (1963) graph the location of their event-stages
against percent of the total developmental period for S. gregaria, and
Wardhaugh (1978) does the same for Chortoicetes. Chapman and Whitham
(1968) and Wardhaugh (1978) have compiled tables showing the percent of
development indicated by events in their staging systems, and in several other
systems. Tables correlating homologous stages in most grasshopper embryogenesis event-staging systems have been prepared by Chapman & Whitham
(1968) and by Micciarelli-Sbrenna (1969). Age-staging and event-staging are
correlated by Shulov & Pener (1959), Salzen (1960), Shulov & Pener (1963),
Van Horn (1967), Chapman & Whitham (1968) and Shulov (1970) for several
genera.
Percent-stages described for one temperature or developmental rate in a
Quantitative staging of embryonic development of Schistocerca
71
given species should be accurate for other temperatures or developmental
rates. Shulov & Pener (1963) incubated batches of S. gregaria embryos at two
different temperatures resulting in embryogenesis times of about 17 and about
50 days. They event-staged embryos developing at these markedly different
rates and graphed stage against percent of total developmental time for the two
temperatures. The two curves were overlapping throughout the whole period of
development, showing that all stages were compressed or expanded proportionally according to developmental rate. This result has been confirmed for
Locusta (Chapman & Whitham, 1968), Schistocerca (Tyrer, 1970) and
Chortoicetes (Wardhaugh, 1978). Therefore, percent-staging should be
independent of developmental rate.
How applicable would these S. nitens percent-stages be to other species of
grasshoppers? They will be inappropriate for species lacking continuous
development (diapausing). Among non-diapausing species, Chapman &
Whitham (1968) have made an extensive comparison of the percentage of
developmental time required to reach a comparable morphological stage for all
of the grasshoppers whose embryogenesis has been carefully described. While
there appear to be some real differences of small magnitude, they conclude that
in general all the non-diapausing species show 'remarkable uniformity' in the
percentage of time taken to reach a given stage. Consequently, major adjustments
would probably not be necessary to match the S. nitens stages to those of other
species.
We have placed our stages at 5 % intervals through embryogenesis. What
percentage error can be expected in the accuracy with which the described
stages match ideal 5 % stages ? The maximum cumulative error which would
allow placement to the nearest 5 % would be 2-5 %, or 12 h at the end of a 20-day
embryogenesis. There are several sources of error. The first is the accuracy with
which the elapsed time of development is known; since egg deposition, when
fertilization occurs, and hatching were directly observed (Materials and
Methods), the maximum error introduced here is 0-75 h (±0-16 %). A second
source of error is the deviation of developmental time of a pod from exactly
20 days. In pod-H, all normally hatching individuals appeared within ± 5 h of
20 days (±1-04 %). Several other pods hatched with slightly longer or shorter
developmental times. Examination of embryos from this set of pods is the basis
of our estimation of the appearance of true 20-day embryos, and of the difference
in appearance introduced by slight deviation from the 20-day period. A third
error is that introduced by asynchrony among embryos developing in the same
pod. In S. nitens, this error is often less than ±0-5 % of total developmental
time (Fig. 1). A similar degree of synchrony has been well documented in other
species of grasshoppers (Bodine, 1925; Slifer, 1932a; Salzen, 1960; Shulov &
Pener, 1963; Shulov, 1970; Tyrer, 1970; Wardhaugh, 1978). In S. gregaria,
Tyrer (1970) reports that for 12 pods examined, 49 % of individuals hatched
within 2 h of the first hatchling, and 82 % within 3 h. If our stage descriptions
72
D. BENTLEY AND OTHERS
were based on individuals expressing errors of maximum size and the same sign,
the cumulative error would be about ± 1-7 %. However, the morphs described
were seen repeatedly, making it highly probable that they were near the mean,
and not near the error extremes. Consequently, the stage descriptions should lie
well within ± 1 % of the ideal 5 % stages (note that the types of errors discussed
here are those involved in constructing the staging system, and are not a concern
in employing it).
We thank Drs Corey S. Goodman and C. M. Bate for criticism of the manuscript. Support
provided by NSF Grant BNS75-03450 and NIH Grant NS-9074-09.
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