Quantitative stagin ogf embryonic developmen otf the ... · subdivision of embryogenesis into equal...

J. Embryol. exp. Morph. Vol. 54, pp. 47-74, 1979 4 7Printed in Great Britain © Company of Biologists Limited 1979

Quantitative staging of embryonic development ofthe 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 completeembryogenesis time, (3) characterization of stages by light micrographs (and descriptivetext), and (4) illustration of stages at the egg, embryo, and limb levels of resolution. Advantagesof a percent-system include communicability, flexibility in temporal resolution, accurateassignment of elapsed time in developmental processes, and uniform coverage of the periodof 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 typesduring 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 approachesare feasible has made grasshopper embryogenesis particularly attractive foranalysing many problems in developmental neurobiology and developmentalbiology in general. To accurately characterize the time course of developmentalevents, it is necessary to have a precise, rapid staging system, applicable tounstained, living material and covering the entire period of embryogenesis.Such a staging has not been available.

There is an extensive literature on grasshopper embryogenesis extendingfrom the mid-nineteenth century (Packard, 1883; Wheeler, 1893; Slifer, 1932a;Roonwal, 1936; Johannsen & Butt, 1941; Anderson, 1972). Several systems forstaging development have been described (Table 3). Although various featuresof these systems are excellent, no single one covers the entire course of embryo-genesis 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 byeasily observable changes in external morphology or orientation of the embryo;as these events are not distributed uniformly throughout development, relativelylong, uncharacterized periods occur in these systems.

In this paper, we present a new staging system. Its main features are: (1) thestages evenly subdivide the period of development; (2) stages are expressed as apercentage of total developmental time; (3) stages are illustrated by lightphotomicrographs; (4) stages are illustrated at three levels of increasing resolu-tion (egg; embryo; limb).

MATERIALS AND METHODS

The experimental animals were Schistocerca nitens, initially captured in 1963and maintained in culture for approximately 50 generations. The animals wereraised 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 wheat-germ and dry dog-food. Generation time was 10-12 weeks.

Egg-pods were deposited in 6 cm diameter/10 cm high paper cups containingcleaned no. 1 sand moistened by 15 % water by weight. Cups were capped duringincubation. Pods contained from 25 to 100 eggs. While pods used for maintain-ing the culture could be left undisturbed, those intended for experiments hadto 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 ofexposed eggs, and covered with damp cotton; alternatively, the pod was com-pletely dispersed and eggs were individually washed in distilled water and placedseparately 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 develop-mental time, and further, to place the stages at equal intervals throughoutembryogenesis. To accomplish this, it was necessary to have a group of syn-chronously developing eggs whose total developmental time could be accuratelypredicted. Sample eggs could then be withdrawn from this group at equalintervals (5 % of total developmental time in this case) and described.

Eggs within the same pod formed our synchronous groups. The total develop-mental time of each egg was determined with an accuracy of ± 45 min. Grass-hopper eggs are fertilized when deposited (McNabb, 1928; Slifer & King, 1934)and completion of a pod takes about 1 h. We selected only pods where depositionwas observed, so the time of fertilization could be determined to ± 30 min.Hatching time was established by continuously observing each pod and countingand removing all the nymphs which hatched within each 30 min period; thehatching time of each nymph was consequently known to ±15 min. This

Quantitative staging of embryonic development of Schistocerca 49information was obtained from ten pods, and formed the data for a quantitativecharacterization of the synchrony of hatching within pods (Figs. 1, 2).

Five percent staging required the selection of 20 equally spaced observationtimes during the complete period of development. This was done by determiningthe temperature at which the eggs developed in 20 days, and then observing theeggs each day at the time of initial deposition of the pod. The appropriatetemperature was predicted by observing the development of 192 pods attemperatures ranging from 30 to 35 °C. The actual developmental time of eachof the pods used for staging was determined as described above.

The staging descriptions are designed to be pre-experimental, and thereforeare based on features which can be seen in unstained, living embryos with adissecting microscope. Correspondingly, all photomicrographs were made witha dissecting microscope (Wild M5A; a few additional features which can beseen in simple squashes in a compound microscope are noted). A text descriptionof 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'tremove it (longer exposure clears better but causes osmotic changes which alterthe shape of the embryo). Cleared eggs were photographed in dark-fieldillumination; embryos were photographed in transmitted illumination or, afterthey became opaque, in incident illumination. All colors are described fromincident illumination.

The saline in which embryos were examined comprised NaCl 140 mM,KC1 5mM, CaCl2.2H2O 4mM, MgSO4.7H2O 2mM, TES 2 HIM, dextrose55-65 mM, pH 7-2, osmolarity; 310-325 milliosmol/kg. High osmolarity wascrucial to maintaining physiological condition and had to be adjusted for age ofthe embryo (Carlson, 1961; Grellet, 1968). It was altered by varying dextroseconcentration to maximize heart rate, peristalsis rate, neuroblast mitosisfrequency or, in early stages, to prevent shrinking or swelling of the amnioticcavity.

The final staging is based upon precisely timed observation of eggs fromeight pods. At each observation period, descriptions of several eggs from eachpod were made; for three pods, photographs of several eggs were made eachday. 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 ofsynchrony can be expressed by calculating the percentage of eggs which hatchedwithin a period equal to 1 % of total developmental time (Fig. 1). In three of four


10

•3 40

30

20

10 -

Pod Cn = 507o= 100

Pod I)

'.'o= 100

19 20 19

Developmental time (days)2G

Fig. 1. Hatching synchrony of eggs within each of four pods (A, B, C, D; incubatedon sand), n = the number of eggs hatching from each pod; % = the percentage ofhatching eggs from each pod which hatched within a period equal to 1 % of thetotal 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; incubatedon filter paper), n = the number of eggs hatching from each pod; % = the per-centage 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 werewetted with cool water at day 19; all of the remaining eggs hatched within the 9 hperiod shown, but the exact time of hatching was not noted.

pods, all the eggs hatched within this period. This degree of synchrony indicatesthat the time of hatching of a subset of eggs from a pod is an accurate estimateof when eggs removed for staging descriptions would have hatched if they hadbeen left undisturbed. Therefore, the percentage of developmental timeexperienced by embryos withdrawn for staging can be estimated within an erroro f l % .

An additional problem is the degree to which synchronous hatching indicatessynchronous development. Mechanisms, such as pheromonal or mechanicalstimulation, might be present which initiate simultaneous hatching in eggs thatare actually at slightly different stages of development. To evaluate this factor,


Fig. 3. Developmental synchrony between eggs from the same pod at different stagesin embryogenesis. Metathoracic limbs of four embryos from the same pod areshown at 55 % development and at 90 % development. The degree of similaritybetween these limbs is representative of the range of variation normally encounteredand indicates that synchrony falls well within the 5 % range throughout embryo-genesis. Transmitted illumination.


Table 1. Features of 5 % developmental stages

Stage Characteristics (distinguishing from previous stage)*

0 % Egg light yellow; lipid droplets uniform in size5 % Egg brown; lipid droplets variable in size; energids in posterior yolk

10 % Disc-shaped blastoderm; energids throughout yolk15 % Embryo cephalized; amnion present; early yolk cleavage20 % Primary segmentation; embryo post-anatrepsis; yolk cleavage about two thirds

length of egg25 % Segmentation to third abdominal segment; neuroblasts visible; leg rudiments

larger than mouthpart rudiments30 % Embryo fully segmented; eye region delaminated; rudiments on first two

abdominal segments35 % Rudiments on all abdominal segments; primary cuticle visible; proctodeum in

tenth abdominal segment40 % Separation of femur and tibia in metathorax; embryo half length of egg;

proctodeum in ninth abdominal segment45 % Separation of tibia and tarsus in metathorax; katatrepsis initiated; proctodeum

in eighth abdominal segment50 % Pigmentation of eye-plate; embryo post-katatrepsis; metathoracic tibia parallel

to femur55 % Antennal furrows; metathoracic tibia reaches base of femur; herringbone array

of extensor tibia muscle fibers60 % White line on eye-plate; rotation of embryo; embryo more than three quarters

length of egg65 % Double curvature of metathoracic tibia; leg twitching; synchronous contraction

of median sinus70 % Metathoracic tibia straightened; embryo pale green; white line close to anterior

margin of eye75 % Longitudinal rows of brown pigment spots line metathoracic femur; brown

pigment on dorsal, caudal midline80 % Brown pigment spots on all legs; apolysis of second embryonic cuticle completed85 % Clearing of femoral crescent; embryo bright green; vertical eye stripes90 % Tar sal claws are black; brown pigmentation covers compound eye; transverse,

dark-green stripes on metathoracic femur95 % Black hairs on antennae; integument opaque white; dark blue color within

antennae and metathoracic legs100 % Egg hatches; in saline, embryos near 100 % begin peristaltic contractions

* Italics indicate most unequivocal feature.

we examined groups of embryos from the same pod at various times in develop-ment. Inspection of rapidly changing features, such as the degree of differentia-tion of the metathoracic limb (Fig. 3), indicated that most embryos within a podwere at a very similar stage of development. Occasional out-of-step embryoswere encountered. These were almost invariably behind their pod-mates,suggesting that they may have been moribund individuals which would havefailed to hatch. Significant variability in the numbers of such individualsoccurred between pods.

We observed pods which hatched in total developmental times ranging fromabout 19-5 to 20-5 days. This gave a good sampling of the developmental rates


surrounding and including 20 days. Daily sampling at these rates provided thebasis for our characterization of 5 % developmental stages. The photomicro-graphs (Figs. 3-9) were made from pods characterized in Fig. 2. Key featuresdelineating each 5% stage are briefly summarized in Table 1. The followingis 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 dueto a darkening of the outer egg envelope or chorion, which is initially transparentand 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 ex-pressed from the egg into saline). As the egg develops they become much moreheterogeneous.

The poles and axes of the egg are assigned by convention with respect tothe orientation of the egg in the maternal ovariole (Mahowald, 1972; Anderson,1972). The posterior pole is marked by a prominent, opaque, cap-like etchingof the chorion (Fig. 4-5). At the base of this cap are the micropyles throughwhich the sperm enter the egg. The anterior pole lies at the opposite, morepointed, end of the egg. Eggs are usually curved. The concave side is dorsaland the convex side ventral (Fig. 4 - 5 0 ) ; however, uncurved or doubly curvedeggs 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 about40 jam in diameter. These are the cleavage energids from which the embryo,embryonic membranes, and yolk cells will develop (Anderson, 1972). Energidscannot be detected through the cleared chorion; they are most easily seen bypuncturing the appropriate part of the egg, squashing the expressed yolk undera coverslip, and viewing in a compound microscope.

10%

At the posterior pole, located toward the ventral side of the egg, is a smallcluster of cells floating on the surface of the yolk droplets (Fig. 6 - 10). Thecluster is a disc-shaped monolayer about 300 /im in diameter and comprises theprimitive 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.


From the outside of a cleared egg, the embryo is now visible on theposterior/ventral surface (Fig. 4 -15) . Its dorsal side is apposed to the yolk andthe anterior end faces the anterior pole of the egg.

The embryo has differentiated a widened, anterior, cephalic region (proto-cephalon) extending about half its length (Fig. 6-15). Antennal and pre-antennal segments will arise here. Mouthpart, thoracic, and abdominal segmentswill 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 thedorsal midline forming a second, inner layer. This inner layer extends abouttwo thirds the length of the embryo, from the posterior end to the mid-proto-cephalon. At its anterior end, it expands to form two lobes. The inner layerarises 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 atthe perimeter of the embryo (Fig. 6-15) and covers the entire ventral surface.This membrane and the extra-embryonic membrane (the serosa) arise at aslightly earlier stage by the fusion of amniotic folds (Roonwal, 1936). The serosaeventually forms a sac enclosing the yolk (50 %; Fig. 7 - 55).

The yolk remains a dispersion of variably sized droplets except at the extremeposterior pole where large, oblong cells, from 200 to 300 /*m in length, mark theonset of yolk cleavage. The yolk is gradually ingested by the formation of thesecells 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 dorsalside of the egg, with its anterior end facing the posterior pole and its dorsalsurface apposed to the yolk.

The protocorm has elongated and differentiated into two distinct regionsseparated by a slight flexure and thickening of the embryo (Fig. 6 - 20). Theanterior region contains the presumptive mouthpart tissue, while the posteriorregion 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 posteriorand lateral to the stomodeum mark the antennal rudiments.

The cleavage of the yolk has progressed to approximately two thirds of thelength 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.


25%Mouthpart, thoracic, and the first three abdominal segments are now clearly

delineated (Fig. 6 - 25). The segments in the mouthpart and thoracic regionseach 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 metathoraciclimb bud consists of an inner mass of cuboidal cells surrounded by a rind oftightly packed columnar cells (Fig. 8-25) ; all limb buds appear this waywhen they first differentiate. Anterior to the stomodeum is a small protruber-ance, the presumptive labrum. The antennal rudiments, lateral to the stomo-deum, have enlarged and turned medially. The abdominal segments at this stagelack limb buds.

The mesoderm has become divided into a series of hollow, segmental blocksof tissue in both the mouthpart and thoracic regions. Mesodermal tissue in theabdomen 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, neuro-blasts (Wheeler, 1893; Carlson, 1961; Bate, 19766). In the protocephalon theyare distributed in packets anterior and lateral to the stomodeum, while in theprotocorm 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 elevenabdominal segments possess limb buds (note that the lateral edge of the segmentis easily mistaken for the limb bud, which lies more medially on the ventralsurface). The metathoracic leg has elongated and has a slight, medially directedbend (Fig. 8 - 30). The mesothoracic and prothoracic legs are about the samesize, but smaller than the metathoracic leg. The labial and maxillary rudimentsare also of equal size, and are about twice as long as the mandibular rudiment.

A thin membrane, the provisional dorsal closure, extends across the dorsalsurface of the embryo. This membrane is supplanted later by a true, dorsalectoderm. On the ventral side of the embryo, the amnion is stretched tightlyover the segmental appendages, folding them medially.

The head capsule has two prominent lobes which will eventually be occupiedby the compound eyes. The posterior portion of these lobes has delaminatedinto two layers separated by a space. The outer layer is the eye plate (Roonwal,1937) which comprises the presumptive retina and associated ommatidialstructures; the inner layer will become the distal portion of the optic lobe of thebrain. 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 ofother segments. They are embryonic structures lost at hatching. Limb budsof 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 clearfilm stretched over the appendages and into the opening of the proctodeum.This cuticle is later detached from the epidermis (apolysed) and replaced by asecondary 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 themetathoracic leg. It marks the beginning of differentiation of the claw retractortendon (apodeme), one of three leg tendons (Snodgrass, 1929). Maxillary andlabial limb buds have become trilobed.

A pair of neurons, the pioneer fibers (Bate, 1976 a), can be seen within eachantenna. The cell bodies are located at the tip of the antenna, adjacent to thecolumnar 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 sidesof the midline in segments anterior to the abdomen. They are viewed mosteasily from the dorsal aspect, and comprise early, axonal outgrowths of ventralcord neurons.

The proctodeum has invaginated to the anterior border of the tenth abdominalsegment (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 itsmedial side (this will be the ventral side of the adult leg) which separates thefemoral and tibial regions (Fig. 8 - 40). The claw retractor tendon has extendedfurther proximally from its invagination, and the leg is heavily invested withspindle-shaped cells (probably myoblasts) and nerve fibers. These features alsooccur in the other thoracic and mouthpart appendages.

In the central nervous system, intersegmental fiber bundles extend along theentire length of the embryo. Transverse, intrasegmental nerve fibers can be seenin all segments.

The proctodeum has invaginated to the anterior border of the ninth abdominalsegment (Fig. 6-40) .


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 itsventral surface will appose the ventral side of the egg.

During katatrepsis, the embryo generates a series of rhythmical, metachronalwaves of posterior to anterior contractions. The waves can be seen in clearedeggs, and occur at 10-20 times per minute in 25 °C saline. They continue in lesspronounced form after the completion of katatrepsis.

The metathoracic leg has an additional constriction separating the tibial andtarsal regions (Fig. 8 - 45). Two tendon invaginations are just noticeable on thefemur, the extensor tibia tendon on the lateral aspect and the flexor tibiatendon on the medial (Fig. 8 - 45, 50). The antennae show the onset of seg-mentation, with four thickenings of the outer, columnar rind. The eleventhabdominal appendages, cerci, have broadened into flattened lobes on either sideof the proctodeum (Fig. 6 - 45).

The posterior margin of the eye plate contains spindle-shaped cells extendingacross its full depth. These appear to be retinula cells of the differentiatingommatidia. 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 intersectingthe longitudinal, intersegmental bundles on both sides of the midline.

On either side of the midline, the dorsal surface of the embryo is flecked withwhite spots. These spots are composed of heavily pigmented cells in a tissuelayer which becomes the fat body.

The proctodeum has invaginated to the anterior border of the eighthabdominal 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 theserosa 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 hasdivided 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. Sexualdifferences 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 ofretinula 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 59region occurs at the intersection of the fiber bundles. This region is the incipientneuropil of the embryonic ganglia.

There is marked apolysis of the primary embryonic cuticle, with the secondarycuticle forming underneath (Mueller, 1963; Micciarelli & Sbrenna, 1972).

The proctodeum has invaginated to the anterior border of the seventhabdominal 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 distanceto the anterior pole (Fig. 5 - 55). The amnion now closes the dorsal surfaceup to the prothorax (Fig. 7 - 55). Yolk is continuous from the serosal sacthrough the open cervical dorsum into the midgut of the embryo. The yellowmidgut yolk is encased by transparent fat-body tissue containing a profusion ofwhite cells (Fig. 7 - 55, 60).

A clear band along the dorsal midline of the embryo demarcates the medianblood sinus, antecedent to the heart. Peristaltic, anteriorly directed constrictionsof 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 tipof the leg has a longitudinal furrow marking the tarsal claws. Well-differentiatedmuscle tissue is visible in transmitted light. A distinctive, herringbone array ofmuscle fibers, the incipient extensor tibia muscle (135a and b; Snodgrass, 1929;Fig. 8 - 55), occurs along the dorsal (previously medial; 45 %) tendon of themetathoracic femur. Localized muscle fiber contractions can be seen throughthe cuticle, but the leg does not twitch noticeably.

The genital appendages (on the eighth and ninth abdominal segments of thefemale and the ninth and tenth of the male) shift toward the ventral midline(stage 3; Karandikar, 1942). No other abdominal limb rudiments remain betweenthe pleuropodia and the cerci. Pronounced furrows in the epidermis divide theantennae into annular segments (Fig. 7 - 55).

A brick-red band starts at the posterior margin of the compound eye andextends forward one fourth of its width (Fig. 7 - 55). This band comprises both asuperficial and a deep layer of the same pigment. Rows of facets line the surfaceof 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 adultommatidium, there are red-brown pigments present in both the photoreceptorsand in two layers of pigment cells that line the outside of each ommatidialcartridge; Roonwal, 1947).

EMB 54


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 inthe completion of expansion and in the amount of space left; about a third ofour embryos did not finish this process until the next stage.

Expansion is accompanied by a 180° rotation of the embryo about its longi-tudinal axis (Slifer, 19326; Bodenheimer & Shulov, 1951; Jones, 1956), leavingits ventral surface adjacent to the dorsal (concave) side of the egg (Fig. 5 - 60).This orientation is maintained for the remainder of embryogenesis. Rotationof 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 protruberanceof degenerating serosal cells (dorsal organ; Wheeler, 1893) which sinks intothe midgut to allow the completion of dorsal closure. After closure, a pairof bilateral bladders, the cervical ampullae, lie between the head and the deeplywrinkled pronotum. They function during hatching and do not persist into thefirst instar (Bernays, 1971).

Genital rudiments of the ninth abdominal segment have partially fused alongthe ventral midline in both sexes. A pair of narrow, flattened rudiments arebarely distinguishable at the posterior margin of the female's eighth abdominalsegment, and the tenth segment rudiments of the male have completely disap-peared underneath the fused ninth pair. The cercus has elongated into a conewhich lies folded beneath the abdomen (Edwards & Chen, 1979).

A white line extends dorso-ventrally along the anterior margin of the eyeplate (Fig. 7 - 65) which is about halfway across the presumptive compoundeye. An unpigmented zone divides this white line from the parallel posteriorband of red pigment. White pigment cells in each ocellus begin to coalesceinto a disc fronted by a lens primordium.

65%

A narrow space persists between the embryo's head and the anterior pole ofthe 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, andthe distal half gradually curves back toward it. This morphological charac-teristic is more reliable than behavioral features for identification of the stage(note: transitional forms obviously occur in the appearance and disappearanceof this and other features; therefore it is important to rely also upon moresubtle changes in the balance of form, such as the relative size of the abdomenand metathoracic leg, which can be apprehended by studying photographs ofthe different stages).

The legs begin to twitch. These periodic jerks are easily distinguished from

Quantitative staging of embryonic development of Schistocerca 61the smooth limb displacement accompanying bodywall peristalsis. Individuallimbs extend rhythmically at one or more joints, but there is no apparent co-ordination of frequency or phase between limbs. Limb movements are notevident until the embryo is removed from the egg.

Peristaltic waves are replaced by simultaneous contraction along the entirelength of the median sinus (Nelson, 1931). Apparently these contractionsrepresent the beginning of normal heartbeat (Roonwal, 1937, described theformation of a definitive heart wall within this sinus at an equivalent stage inLocustd). White fat cells begin to appear beneath the transparent heart tube,reflecting its detachment from the midgut and the subsequent intrusion of thefat body.

The labia have shifted medially and fused at the base. Other mouthpartsremain 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 theprevious stage (Fig. 9 - 70). The tibia will not become perfectly straight untilafter 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 deeplayer, forming a speckled, light-red band along its leading edge. A colorless zonestill separates the red region from the anterior white band (Fig. 7 - 70). Thisband has also moved anteriorly and approaches the frontal margin of the eye.

In some individuals, brown pigment fringes the cuticular sleeve envelopingthe mandible rudiment (this feature should not be mistaken for the extensivepigmentation of the mandible teeth that occurs at 90 % development).

75%

This stage is distinguishable by the appearance of longitudinal rows ofbrown spots on the metathoracic femur (Fig. 7 - 7 5 ; 9 - 85). The rows containabout ten spots each, and mark incipient ridges along the dorsal and ventraledges 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 appearanceunder incident illumination.

White teeth begin to form on the medial edge of the mandible rudiment,within the cuticular sleeve.

5-2


80%

For most embryos, the head is tightly pressed into the anterior end of theegg (Fig. 5 - 80).

Brown pigment spots are present on the femur and tibia of every leg, but noton the head or thoracic body-wall. A faint, brown dorsal midline extendsforward to the mesothorax.

The second embryonic cuticle separates from the underlying epidermis overthe entire body surface, and the third cuticle begins to form. The second cuticleremains intact throughout embryogenesis and is shed soon after hatching(Bernays, 1972). The third cuticle will be the cuticle of the first instar. Apolysisof the second cuticle has been described from histological sections for S. gregariaresembling our stages 70-75 (Micciarelli & Sbrenna, 1972), and can also beseen in S. nitens embryos of this stage with a compound microscope.

The brown pigment band of the compound eye may encroach upon theposterior edge of the dorsal spot, a smooth, indistinctly faceted ellipse the size ofan ocellus situated at the dorsal margin of the eye.

85%

The entire embryo is bright green in color, although the midgut yolk stilllends 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 metathoracicfemur (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 fillsthe posterior half of the dorsal spot. The brown color remains dark within theboundary of this structure, but pales elsewhere except for two vertical stripeswhich persist within the light-brown region (Fig. 7 - 85, 90). These stripes willdisappear later and are not antecedent to the postembryonic striations describedby Roonwal (1947). A circular, black image known as the pseudopupil appearsto lie beneath the eye surface (Fig. 7 - 85, 90). The pseudopupil is not a structurebut an optical illusion manifest in the organization of the eye (Horridge, 1977).

Cereal sensory hairs have grown into the empty tip of the cuticular sheathin some individuals (they are not pigmented and must be viewed in transmittedlight). The presence of sensilla demonstrates the deposition of a definitivefirst instar cuticle on the surface of the epidermis (Edwards & Chen, 1979).

90%

Three broad transverse stripes of dark green appear on the metathoracicfemur (Figs. 7 - 9 0 ; 9-90) , accentuating the herringbone pattern on the

Quantitative staging of embryonic development of Schistocerca 63external surface. The pattern reflects a double oblique array of low ridgesoverlying the insertions of the extensor tibia muscle fibers on to the inner side ofthe cuticle. Brief cuticle indentations occur sporadically within the insertionarea. They are reminiscent of the fiber twitches seen at stage 55, and implythat 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 iscontiguous 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 hairsnow appear black under incident illumination. The teeth of the mandible, andsometimes those of maxilla, are a lustrous brown (Fig. 7 - 90). Brown spottinghas progressed to the antennae, tarsi, palps, abdominal tergites, and occipitalarea of the head.

95%

The embryonic integument turns opaque white on much of the head andlimbs and on small patches of the body. Brown and black markings areprominent against this background. The degree of white coloration on theabdomen varies greatly between individuals, so that the heart may be eitherexposed or obscured.

The teeth of both the mandible (Fig. 7 - 95) and maxilla darken to black atthe tips, as do the tibial spines which are pressed flat against the legs by theembryonic 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 tintis pronounced around the femoro-tibial articulation, beneath the marginalridges of the femur, and in the proximal segments of the antennae.

Eye color progressively pales. The vertical stripes have faded, and the eye isan essentially uniform shade of brown except for the dark dorsal spot.

In most individuals, the spontaneous cuticle indentations seen in the previousstage are no longer apparent.

100%

No external morphological features unequivocally distinguish this stage fromthe previous. The most distinctive difference between the two is their respectivecompetence to hatch. Hatching is effected by a series of powerful, anteriorlydirected peristaltic contractions of the body wall which rupture the egg andliberate the embryo still encased in the second embryonic cuticle (vermiformlarva; Bernays, 1971). Hatching competence was quantified by testing embryosat various stages between 90 % and 100 % from three pods (percentage develop-ment was calculated from the median length of embryogenesis for the remainderof each pod). Embryos were released from the egg under 24+1 °C saline; if


Table 2. Hatching competence of maturing embryos

90 % stage90-92-5

95 % stage

92-5-95 95-97-5100 % stage

97-5-100

Number testedNumber hatching"Percent hatching

00%

152

13%

122

17%

201680%

* Showing rhythmical sequences of peristaltic waves (see text).

Fig. 4. Appearance of live eggs at 5 % developmental intervals during the first half ofembryogenesis (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 withinegg; 50, compound eye. Dark-field illumination.


Fig. 5. Appearance of live eggs at 5 % developmental intervals during the secondhalf of embryogenesis (eggs partially cleared in sodium hypochlorite). 55-60, notethat the convex side of the egg has changed from ventral (55) to dorsal (60); thisreflects the 180° rotation of the embryo within the egg. Dark-field illumination.

contractions did not appear, they could sometimes be elicited by brushing thelegs with forceps. Only embryos with the 95 % - 100 % morphology producedrhythmical sequences of peristaltic waves (Table 2), and the behavior occurredmuch more reliably in embryos near 100 %.

Newly hatched larvae normally must dig upward to reach the surface of theground (Bernays, 1971). Larvae hatching on the surface begin molting theembryonic cuticle (first ecdysis) within 10 min. In the first ecdysis, the cuticularenvelope is split along the dorsal midline and the first instar nymph emerges.Emergence is produced by a complex sequence of actions very similar to thatdescribed for S. gregaria (Bernays, 1972). As the embryonic cuticle is sloughed,numerous black and unpigmented hairs spring erect, the cervical ampullaedeflate, and the mouthparts close medially for the first time. Following ecdysis,


Fig. 6. Appearance of live embryos at 5 % developmental intervals during the firsthalf of embryogenesis. 5, site in yolk droplets where embryonic disc will appear;10 (arrows), embryonic disc. 35, note amnion (membrane) spread out around theanterior end of the embryo. Transmitted illumination.

the nymph is quiescent for ten to fifteen minutes before taking its initialsteps.

The first instar nymph is bright green with the same brown and black markingsas the embryo. Blue pigment persists within the antennae, but fades from themetathoracic femur at the time of hatching. Faint vestiges of the embryoniceye stripes may be visible even after the broad, dark first instar stripe begins togrow down from the dorsal spot (Roonwal, 1947).


Fig. 7. Appearance of live embryos at 5 % developmental intervals during the lasthalf 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 grasshopperembryogenesis. 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 haspassed (percent-staging). For present purposes, age-staging is unsuitablebecause of its inflexibility; it cannot be used when the same species is incubatedat different temperatures, or for different species, or for species with individualvariability in developmental rate. Although the event-staging system may bemore useful for comparing similar stages among widely differing species, we


Fig. 8. Appearance of a metathoracic limb of live embryos at 5 % developmentalintervals from 25 % to 60 % of embryogenesis. Note that there are marked differencesbetween each stage, particularly involving segmentation, flexion, invaginationof apodemes, and differentiation of musculature. Transmitted illumination.


Fig. 9. Appearance of a metathoracic limb of live embryos at 5 % developmentalintervals from 65 % to 100 % of embryogenesis (65-80, transmitted illumination;85-100, incident illumination).

prefer the percentage system for studies of developmental processes for thefollowing reasons: (1) the meaning of a percent-stage is readily apprehended bynon-specialists. This greatly facilitates communication with developmentalbiologists who are not entomologists (in practice, the event-staging systems havebeen essentially species specific). (2) percent-staging allows greater flexibility intemporal resolution. If more temporal detail is required, it is straightforward toextend a 5 % level to a 1 % level of resolution. This kind of modification is acontinuing problem with event-staging systems once the stages have beeninitially erected. (3) percent-staging allows an even distribution of stages (atwhatever level of resolution is required) through the entire developmental


Table 3. Stagings of grasshopper embryogenesis

Genus Reference

Schistocerca Jhingran (1947), Shulov & Pener (1963), Micciarelli-Sbrenna (1969),Tyrer (1970)

Melanoplus Nelson (1931, 1934), Slifer (1932o), Salt (1949), Riegert (1961)Locusta Roonwal (1936, 1937), Shulov & Pener (1959), Salzen (1960)Chortoicetes Wardhaugh (1978)Ornithacris Chapman & Whitham (1968)Pyrgomorpha Chapman & Whitham (1968)Nomadacris Shulov (1970)Locustana Matthee (1951)Euprepocnemis Khalifa (1957)Dociostaurus Bodenheimer & Shulov (1951)Austroicetes Steele (1941)Aulocara Van Horn (1967)Acrida Kucukeksi (1964)

period. The temporal placement of event-stages is dictated by the occurrence ofeasily recognized events and always results in a non-uniform distribution ofstages. Since cellular and sub-cellular processes of great importance in differ-entiation may not be coupled to these easily recognized events, it is better tohave a staging system which does not leave gaps in the complete course ofembryogenesis. (4) percent-staging permits accurate assignment of elapsed time indevelopmental processes. Using event-staging, it is possible to order observa-tions but not to discuss the real or relative durations of processes; it is meaning-less to consider elapsed time between two observations unless both are madein the same animal. A percent system makes it possible to construct the develop-mental history of a process, using data collected from many individuals, withrecognition of the time between succeeding steps.

The percent-staging system presented here can be correlated with previouslydescribed systems (Table 3). Tyrer (1969, 1970) employed a percent-stagingsystem at a 10 % level of resolution for the last 40 % of embryogenesis ofS. gregaria. Shulov & Pener (1963) graph the location of their event-stagesagainst percent of the total developmental period for S. gregaria, andWardhaugh (1978) does the same for Chortoicetes. Chapman and Whitham(1968) and Wardhaugh (1978) have compiled tables showing the percent ofdevelopment indicated by events in their staging systems, and in several othersystems. Tables correlating homologous stages in most grasshopper embryo-genesis event-staging systems have been prepared by Chapman & Whitham(1968) and by Micciarelli-Sbrenna (1969). Age-staging and event-staging arecorrelated by Shulov & Pener (1959), Salzen (1960), Shulov & Pener (1963),Van Horn (1967), Chapman & Whitham (1968) and Shulov (1970) for severalgenera.

Percent-stages described for one temperature or developmental rate in a

Quantitative staging of embryonic development of Schistocerca 71given species should be accurate for other temperatures or developmentalrates. Shulov & Pener (1963) incubated batches of S. gregaria embryos at twodifferent temperatures resulting in embryogenesis times of about 17 and about50 days. They event-staged embryos developing at these markedly differentrates and graphed stage against percent of total developmental time for the twotemperatures. The two curves were overlapping throughout the whole period ofdevelopment, showing that all stages were compressed or expanded propor-tionally according to developmental rate. This result has been confirmed forLocusta (Chapman & Whitham, 1968), Schistocerca (Tyrer, 1970) andChortoicetes (Wardhaugh, 1978). Therefore, percent-staging should beindependent of developmental rate.

How applicable would these S. nitens percent-stages be to other species ofgrasshoppers? They will be inappropriate for species lacking continuousdevelopment (diapausing). Among non-diapausing species, Chapman &Whitham (1968) have made an extensive comparison of the percentage ofdevelopmental time required to reach a comparable morphological stage for allof the grasshoppers whose embryogenesis has been carefully described. Whilethere appear to be some real differences of small magnitude, they conclude thatin general all the non-diapausing species show 'remarkable uniformity' in thepercentage of time taken to reach a given stage. Consequently, major adjustmentswould probably not be necessary to match the S. nitens stages to those of otherspecies.

We have placed our stages at 5 % intervals through embryogenesis. Whatpercentage error can be expected in the accuracy with which the describedstages match ideal 5 % stages ? The maximum cumulative error which wouldallow placement to the nearest 5 % would be 2-5 %, or 12 h at the end of a 20-dayembryogenesis. There are several sources of error. The first is the accuracy withwhich the elapsed time of development is known; since egg deposition, whenfertilization occurs, and hatching were directly observed (Materials andMethods), the maximum error introduced here is 0-75 h (±0-16 %). A secondsource of error is the deviation of developmental time of a pod from exactly20 days. In pod-H, all normally hatching individuals appeared within ± 5 h of20 days (±1-04 %). Several other pods hatched with slightly longer or shorterdevelopmental times. Examination of embryos from this set of pods is the basisof our estimation of the appearance of true 20-day embryos, and of the differencein appearance introduced by slight deviation from the 20-day period. A thirderror is that introduced by asynchrony among embryos developing in the samepod. In S. nitens, this error is often less than ±0-5 % of total developmentaltime (Fig. 1). A similar degree of synchrony has been well documented in otherspecies 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 hatchedwithin 2 h of the first hatchling, and 82 % within 3 h. If our stage descriptions


were based on individuals expressing errors of maximum size and the same sign,the cumulative error would be about ± 1-7 %. However, the morphs describedwere seen repeatedly, making it highly probable that they were near the mean,and not near the error extremes. Consequently, the stage descriptions should liewell within ± 1 % of the ideal 5 % stages (note that the types of errors discussedhere are those involved in constructing the staging system, and are not a concernin employing it).

We thank Drs Corey S. Goodman and C. M. Bate for criticism of the manuscript. Supportprovided by NSF Grant BNS75-03450 and NIH Grant NS-9074-09.

REFERENCESANDERSON, D. T. (1972). The development of hemimetabolous insects. In Developmental

Systems. Insects, vol. I (ed. S. J. Counce & C. H. Waddington), pp. 96-165. New York,London: Academic Press.

BATE, C. M. (1976a). Pioneer neurons in an insect embryo. Nature, Lond. 260, 54-55.BATE, C. M. (19766). Embryogenesis of an insect nervous system. 1. A map of the thoracic

and abdominal neuroblasts in Locusta migratoria. J. Embryol. exp. Morph. 35, 107-123.BENTLEY, D. & TOROIAN-RAYMOND, A. (1979). Embryonic development of the dendritic

arborization of an identified insect interneuron (unpublished).BERNAYS, E. A. (1971). The vermiform larva of Schistocerca gregaria (Forskal): form and

activity (Insecta, Orthoptera). Z. Morph. Okol Tiere 70, 183-200.BERNAYS, E. A. (1972). The intermediate moult (first ecdysis) of Schistocerca gregaria

(Forskal) (Insecta, Orthoptera). Z. Morph. Okol Tiere 71, 160-179.BODENHEIMER, F. S. & SHULOV, A. (1951). Egg development and diapause in the Moroccan

locust. Bull. Res. Coun. Israel. 1, 59-75.BODINE, J.H. (1925). Effect of temperature on rate of embryonic development of certain

Orthoptera. J. exp. Zool. 42, 91-109.CARLSON, J. G. (1961). The grasshopper neuroblast culture technique and its value in radio-

biological studies. Ann. N.Y. Acad. Sci. 95, 932-941.CHAPMAN, R. F. & WHITMAN, F. (1968). The external morphogenesis of grasshopper embryos.

Proc. Roy. ent. Soc. Lond. {A) 43, 161-169.EDWARDS, J. S. & CHEN, S. (1979). Embryonic development of an insect sensory system, the

abdominal cerci of Acheta domesticus. Wilhelm Roux' Arch. devl. Biol. 186, 151-178.GOODMAN, C. S., O'SHEA, M., MCCAMAN, R. & SPITZER, N. C. (1979). Embryonic develop-

ment of identified neurons: temporal pattern of morphological and biochemical differentia-tion. Science. 204, 1219-1222.

GOODMAN, C. S. & SPITZER, N. C. (1979). Embryonic development of identified neurons:differentiation from neuroblast to neuron. Nature, Lond. 280, 208-214.

GRELLET, P. (1968). Milieu de culture pour embryons de gryllides. / . Insect Physiol. 14,1735-1761.

HORRIDGE, G. A. (1977). The compound eye of insects. Sci. Amer. 237, 108-120.JHINGRAN, V. G. (1947). Early embryology of the desert locust, Schistocerca gregaria

(Forskal) (Orthoptera, Acrididae). Rec. Indian Mus. 45, 181-200.JOHANNSEN, O. A. & BUTT, F. H. (1941). Embryology of Insects and Myriapods. New York,

London: McGraw-Hill.JONES, B. M. (1956). Endocrine activity during insect embryogenesis. Function of the ventral

head glands in locust embryos {Locustana pardalina and Locusta migratoria, Orthoptera)./ . exp. Biol. 33, 174-185.

KARANDIKAR, K. R. (1942). External structures of the desert locust {Schistocerca gregaria,Forsk). / . Univ. Bombay 11:3B, 1-29.

Quantitative staging of embryonic development of Schistocerca 73KHALIFA, A. (1957). The development of eggs of some Egyptian species of grasshoppers,

with special reference to the incidence of diapause in the eggs in Euprepocnemis ploransCharp. Bull. Soc. Entomol. Egypt. 41, 299-330.

KUCUKEKSI, S. (1964). Contribution to the study of egg development in Acrida bicolor(Thunb.) (Orthoptera, Acrididae). Communs. Fac. Sci. Univ. Ankara (C) 13, 131-178.

MAHOWALD, A. P. (1972). Oogenesis. In Developmental Systems. Insects, vol. i (ed. S. J.Counce & C. H. Waddington), pp. 1-48. New York, London: Academic Press.

MATTHEE, J. J. (195.1). The structure and physiology of the egg of Locustana pardalina(Walk.). Sclent. Bull. Dep. Agric. S. Afr. 316, 1-83.

MCNABB, J.W. (1928). A study of the chromosomes in meiosis, fertilization, and cleavagein the grasshopper egg (Orthoptera). / . Morph. Physiol. 45, 47-93.

MICCIARELLT-SBRENNA, A. (1969). Gli stadi normali di sviluppo degli embrioni di Schistocercagregaria Forskal (Orthoptera, Acrididae). Boll. Zoo I. 36, 77-95.

MICCIARELLI, A. S. & SBRENNA, G. (1972). The embryonic apolyses of Schistocerca gregaria(Orthoptera). / . Insect Physiol. 18, 1027-1037.

MUELLER, N. S. (1963). An experimental analysis of molting in embryos of Melanoplusdifferentialis. Devi Biol. 8, 222-240.

NELSON, O. E. (1931). Life cycle, sex differentiation, and testis development in Melanoplusdifferentialis (Acrididae, Orthoptera). / . Morph. Physiol. 51, 467-525.

NELSON, O. E. (1934). Segregation of the germ cells in the grasshopper, Melanoplusdifferentialis. / . Morph. 55, 545-575.

PACKARD, A. S. (1883). The embryological development of the locust. Third Report of theU.S. Entomol. Comm. pp. 263-285.

RIEGERT, P. W. (1961). Embryological development of a non-diapausing form of Melanoplusbilituratus Walker (Orthoptera, Acrididae). Can. J. Zool. 39, 491-494.

ROONWAL, M. L. (1936). Studies on the embryology of the African Migratory Locust,Locusta migratoria migratorioides R. and F. I. The early development, with a new theoryof multi-phased gastrulation among insects. Phil. Trans. R. Soc. Lond. B 226, 391-421.

ROONWAL, M. L. (1937). Studies on the embryology of the African Migratory Locust,Locusta migratoria migratorioides R. and F. (Orthoptera, Acrididae). Phil. Trans. R. Soc.Lond. B 227, 175-244.

ROONWAL, M. L. (1947). Variation and structure of the eyes in the desert locust, Schistocercagregaria (Forskal). Proc. R. Soc. Lond. B 134, 245-272.

SALT, R. W. (1949). A key to the embryological development of Melanoplus bivittatus (Say),M. mexicanus mexicanus (Sauss.), and M. packardii (Scudder). Can. J. Research. B 27,233-235.

SALZEN, E. A. (1960). The growth of the locust embryo. / . Embryol. exp. Morph. 8, 139-162.SHARAN, R. K. (1958). The embryonic cuticle of Locustana'pardalina. Annls Zoology 3,1-8.SHULOV, A. (1970). The development of eggs of the Red Locust, Nomadacris septemfasciata

(Serv.), and the African Migratory Locust, Locusta migratoria migratorioides (R. and F.),and its interruption under particular conditions of humidity. Anti-Locust Bull. 48, 1-22.

SHULOV, A. & PENER, M. P. (1959). A contribution to knowledge of the development of theegg of Locusta migratoria migratoriodes. Locusta 6, 73-88.

SHULOV, A. S. & PENER, M. P. (1963). Studies on the development of eggs of the desertlocust {Schistocerca gregaria Forskal) and its interruption under certain conditions ofhumidity. Anti-Locust Bull. 41, 1-59.

SLIFER, E. H. (1932a). Insect development. IV. External morphology of grasshopper embryosof known age and with a known temperature history. / . Morph. 53, 1-21.

SLIFER, E. H. (19326). Insect development. III. Blastokinesis in the living grasshopper egg.Biol. Zbl. 52, 223-229.

SLIFER, E. H. & KING, R. L. (1934). Insect development. VII. Early stages in the developmentof grasshopper eggs of known age and with a known temperature history. / . Morph. 56,593-601.

SNODGRASS, R. E. (1929). The thoracic mechanism of a grasshopper, and its antecedents.Smithson. misc. Collns. 82, 1-111.

SPITZER, N. C. (1979). Ion channels in development. Ann. Rev. Neurosci. 2, 363-397.


STEELE, H. V. (1941). Some observations on the embryonic development of Austroicetescruciata Sauss. (Acrididae) in the field. Trans Roy. Soc. Sci. Aust. 65, 329-332.

TYRER, N. M. (1969). The time course of contraction and relaxation in embryonic locustmuscle. Nature, Lond. 224, 815-817.

TYRER, N. M. (1970). Quantitative estimation of the stage of embryonic development in thelocust, Schistocerca gregaria. J. Embryol. exp. Morph. 23, 705-718.

VAN HORN, S. N. (1967). Studies on the embryogenesis of Aulocara elliotti (Thomas) (Orthop-tera; Acrididae). External morphogenesis. / . Morph. 120, 83—114.

WARDHAUGH, K. G. (1978). A description of the embryonic stages of the Australian PlagueLocust, Chortoicetes terminifera (Walk.). Acrida 7, 1-9.

WHEELER, W. M. (1893). A contribution to insect embryology. / . Morph. 8, 1-160.

{Received 6 March 1979, revised 4 April 1979)

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