the embryology of dacus tryoni (frogg.) [diptera, trypetidae (= … · the embryology of dacus...

The Embryology of Dacus tryoni (Frogg.)[Diptera, Trypetidae ( = Tephritidae)],

the Queensland Fruit-Flyby D. T. ANDERSON1

From the Zoology Department, University of Sydney

WITH TWO PLATES

INTRODUCTION

THE embryology of the Diptera has been investigated in only a few species andof these, only Drosophila melanogaster and Calliphora erythrocephala have beenstudied in detail. Fragmentary information is available for 17 species of Nema-tocera, several further species of acalyptrate Cyclorrhapha (all Drosophilaspecies), 6 further species of calyptrate Cyclorrhapha, and 2 species of pupi-parous Cyclorrhapha, but little attempt has been made to compare one withanother. The present description of the embryology of the larva of the acalyp-trate Dacus tryoni, the first of a member of the Tephritidae, provides new dataon which a comparative survey of the embryology of dipteran larvae can bebased.

MATERIALS AND METHODS

Material for this study was supplied by Dr. M. A. Bateman of the Joint Unitof Animal Ecology in the Department of Zoology, University of Sydney, fromhis laboratory stocks. Rearing of D. tryoni in the laboratory has been fullydescribed by Bateman (1958) and only a brief outline will be presented here.Adult flies were kept in cages at 25° C, 80 per cent, relative humidity. Whendomes of apple skin, sealed by paranin wax to flat glass plates and pierced bya number of fine holes, were placed in the cages, females settled on them andlaid eggs through the holes into the domes. An oviposition time of 15 minuteswas allowed for each dome, so that the age of eggs subsequently examined wasknown to within ±7£ minutes. After completion of oviposition, the eggs weretransferred by means of a fine brush wetted with distilled water on to squaresof damp filter paper in a Petri dish and left at 25° C, 80 per cent. r.h. untilrequired. In these conditions, hatching of the larva, a typical cyclorrhaphanmaggot, took place approximately 42 hours after oviposition. The few hatched

1 Author's address: Zoology Department, University of Sydney, Australia.[J. Embryo!, exp. Morph. Vol. 10, Part 3, pp. 248-92, September 1962]

EMBRYOLOGY OF THE QUEENSLAND FRUIT-FLY 249

larvae used to complete the final stages of the study were allowed to burrowand feed in carrot medium at the same temperature until required.

From eggs obtained in this way, development was followed from ovipositionto hatching in living embryos and by serial sections. Living embryos wereexamined after removal of the opaque chorion of the egg by immersion for30 seconds in 10 per cent, sodium hypochlorite followed by transfer to distilledwater. The transparent vitelline membrane within the chorion remained intact,protecting the embryo and at the same time permitting observation of changesin external form.

In the preparation of serial sections, other eggs were fixed in aqueous Bouinat boiling-point for 10 minutes, transferred to fresh cold Bouin for 24 hours, thenstored in 70 per cent, alcohol. Fixation in this way facilitated penetration of theegg membranes by the fixative and also caused the membranes to swell andbecome transparent, so that they could easily be dissected off. After passagethrough 95 per cent, alcohol, methyl benzoate and benzene, the embryos wereembedded in paraffin (m.p. 56° C) , sectioned at 8 /x transversely, sagittally, orfrontally and stained with Mayer's haemalum and eosin.

RESULTS

The unfertilized egg

The egg of D. tryoni is elongate, curved, 900-1,150 [i long and 170-210/u, indiameter. One end, the anterior, is drawn out as a pointed papilla, the oppositeend is gently rounded. The antero-posterior axis of the egg is established in theovary and the posterior end emerges first at oviposition. The dorso-ventral axisis also fixed during oogenesis and reflected in the curvature of the egg, its convexsurface being ventral.

The egg is covered by a tough, pearly-white chorion marked externally by aregular lattice of ridges. Immediately within the chorion lies a thin vitellinemembrane and within this again the whitish granular living substance of theoocyte. Both chorion and vitelline membrane have a micropyle at the anteriorpole of the egg (Text-fig. 2B). The oocyte has the characteristic dipteran struc-ture (Text-figs. 1A; 2A), with a thin surface layer of cytoplasm, the periplasm,slightly thickened at the anterior and posterior poles, enclosing a central massof cytoplasm containing abundant yolk spheres of various sizes interspersedwith numerous vacuoles. Within the posterior thickening of the periplasm liesan additional mass of small polar granules having a high affinity for haema-toxylin stains. The cytoplasmic reticulum which forms the walls of the vacuolesand holds the yolk is continuous with the periplasm at the surface. The vacuoleshave a constant distribution and appearance in fixed eggs, being relatively smalland scattered throughout most of the interior, but including a peripheral layerof larger vacuoles absent only at the anterior and posterior ends. Although this

250 D. T. ANDERSON—EMBRYOLOGY OF THE

appearance must to some extent be an artefact, its constancy indicates an under-lying special distribution of certain, as yet unknown, egg constituents.

posterior pole plasm

polar Ngranules

A

oocyte nucleus

anteriorplasm

B

posterior endof qerm band

cleavaqenucleus

periplasm

temporary cephalicfold \ extra-embryonic | u r r S w

extra-embryonic/ectoderm

lateral horn of extra-embryonic ectoderm

proctodaeal /opening /

TEXT-HG. 1. A, oocyte, diagrammatic reconstruction, B, embryo during 5th cleavage, diagrammaticreconstruction, c, 8£-hour embryo, lateral view, D, 12-hour embryo, lateral view.

Close to the anterior pole of the egg lies the oocyte nucleus, surrounded bya halo of yolk-free cytoplasm (Text-fig. 1A). With a diameter of 30 /x and small

QUEENSLAND FRUIT-FLY 251

scattered chromatin granules as its only discernible content in fixed preparations,it is clearly distinguishable from the zygote nucleus resulting from fertilization.

Maturation and fertilization

Just before oviposition, a single sperm enters the micropyle, penetrates thesurface of the oocyte antero-ventrally, and passes into the central region of theanterior end of the egg, pulling in with itself part of the surface periplasm asa sperm track (Text-fig. 2B). Some of this cytoplasm no doubt contributes laterto the cytoplasmic halo of the zygote nucleus.

Maturation of the oocyte is associated with insemination, but the time rela-tions of the two processes and the details of maturation are not yet clear.Immediately after oviposition, the halo of the oocyte nucleus, now containinga number of small scattered chromosomes, lies at the dorsal surface of the eggnear the anterior pole. Within the next 20 minutes a spindle forms and a typicalmaturation division proceeds (Text-fig. 2B). The resulting polar nucleus, em-bedded in the periplasm of the egg, soon disappears. The female pronucleusunites with the male pronucleus and the resulting zygote nucleus, surroundedby a cytoplasmic halo formed by fusion of the haloes of the pronuclei, thenmigrates to the midline of the egg a short distance from the anterior pole(Plate 1, fig. A). In contrast to the oocyte nucleus, the zygote nucleus, formedwithin 30 minutes of oviposition, is 5 /x in diameter and has no distinct chromatingranules, although it shows a weak Feulgen reaction.

In the absence of further evidence it seems likely that the maturation divisionwhich follows oviposition is the second and that the first division is completedbefore the egg is laid. If this is so, the fate of the first polar nucleus has yet to beelucidated.

Cleavage and formation of the blastoderm

Within 30 minutes of its formation the zygote nucleus divides by mitosis andits products divide again in synchrony (Text-fig. 2c; Plate 1, fig. B) to give4 cleavage nuclei, each with its own cytoplasmic halo, lying at the border of theanterior third of the embryo.

Changes in the egg cytoplasm accompany these divisions. Vacuolation is morenoticeable, commensurate perhaps with the manufacture of additional cyto-plasmic haloes at the expense of the cytoplasmic reticulum. An antero-posteriorelongation of the vacuoles of the posterior third of the egg also takes place,indicating a transfer of special substances either towards the posterior pole orfrom the posterior pole forwards (Plate 1, fig. B). Such tendencies become moreplain during the subsequent cleavages giving 8, 16, 32, and 64 nuclei. These, the3rd to 6th mitoses of cleavage, with their accompanying divisions of cytoplasmichaloes and interim periods of nuclear interphase and cytoplasmic accretion, arealso synchronous and occupy the 2nd hour after oviposition. At the same timethe nuclei spread posteriorly through the egg (Text-fig. 1B; Plate 1, fig. C) but


do not invade the periplasm. The rate of division during the 2nd hour, onemitotic wave every 15 minutes, is identical with that of the first two cleavages.

The 3rd hour of development brings a change in nuclear behaviour, followingfirst the synchronous division of 64 into 128 nuclei (7th mitosis of cleavage).

qerminal vesicle

vocuole \^m>, yolk

micropyle

vitel linemembrane

sperm nucleus

sperm track

dorsal suface

maturationdivision

first cleavagemitosis

Banteriorpole plasm

periplasm

cTEXT-FIG. 2. A, frontal section through anterior end of oocyte. B, sagittal section through anterior endof egg fixed 17 minutes after oviposition. c, frontal section through anterior end of egg fixed 40 minutes

after oviposition.

Many nuclei now migrate to the egg surface and enter the periplasm, theircytoplasmic haloes fusing with the latter, while the remainder hold their positionin the yolk as vitellophages. The distinction between surface and vitellophagenuclei thus established is permanent, since no vitellophages subsequently move

Q U E E N S L A N D F R U I T - F L Y 253

TEXT-FIG. 3. A, 3-hour embryo, diagrammatic reconstruction, B, transverse section through middle region of embryo approaching 3 hours (interphase after 9th cleavage), c, transverse section through blastoderm of embryo approaching 5 hours (interphase after 12th cleavage), D , sagittal section through blastoderm of 5-hour embryo, E, transverse section through blastoderm of 6-hour embryo (compare Plate 1, fig. D ) . F, sagittal section through blastoderm of 7-hour embryo (compare Plate 1, fig. E ) .

G, 7^-hour embryo, diagrammatic reconstruction.

to the surface and no surface nuclei, with one exception (see below), re-enter the yolk as vitellophages. Approximately 90 surface nuclei and 38 vitellophage nuclei are separated out, the numbers varying slightly from one egg to another. Nuclear invasion of the surface is simultaneous throughout the entire periplasm. At this stage, when the cytoplasmic haloes are being drawn into the periplasm and


considerable reorganization of the embryo is taking place, the yolky inner massof the egg becomes difficult to fix and generally shows large vacuoles, no doubtfixation artefacts, indicative of an increased fluidity.

By the end of the 3rd hour the nuclei have undergone three further syn-chronous mitoses (Text-fig. 3B) giving 180, 360, then 720 nuclei at the surfaceand 75, 150, then 300 vitellophage nuclei scattered throughout the yolk withtheir cytoplasmic haloes united by the cytoplasmic reticulum both to each otherand to the surface layer (Text-fig. 3A). Division of the vitellophage nuclei appearsto cease in many eggs at this stage, although in a few cases a further wave ofmitosis coinciding with the penultimate mitotic wave at the surface takes placeand the final number of vitellophages becomes approximately 600. The rate ofmitotic division in the 3rd hour of development is sustained at one per 15 minutes.

Towards the end of the 3rd hour, 4 posterior surface nuclei associated withthe polar granules come to lie in cytoplasmic protrusions at the posterior end ofthe egg (Text-fig. 3A). During the next hour of development the protrusions arecut off as 4 pole cells which have the polar granules concentrated wholly withintheir cytoplasm.

In the 4th hour of development, two further synchronous waves of mitotisat the surface, the 11th and 12th of cleavage (Text-fig. 3 c) (rate of divisionhalved), produce approximately 1,440 then 2,880 surface nuclei. The pole cells,however, do not divide in synchrony with the surface nuclei. During the 4th hourthey divide once, becoming 8, and during the 5th hour once again, becoming 16(Text-fig. 4A). The cytoplasm of each cell contains polar granules. The pole-cellgroup as a whole is separated by a small space from the underlying surface ofthe embryo, which in this vicinity is relatively free of nuclei, save for a few,between 2 and 5, within an inwardly projecting cone of surface cytoplasm(Text-fig. 4A). One or two of these nuclei separate off about this time, each witha cytoplasmic halo, as secondary vitellophages. The others remain within thecytoplasmic cone (Text-fig. 4 B, C).

The nuclei at the surface of the embryo undergo a final wave of mitosis, the13th of cleavage, during the 5th hour of development, establishing the definitivenumber of approximately 5,800, tightly packed within the periplasm (Text-figs.3 D ; 4A). The latter is also greatly thickened, the major part of the cytoplasmicreticulum having been drawn into it, leaving the yolk as a central mass of tightlypacked granules with vitellophages scattered among them. The surface layercan now be termed a syncytial blastoderm.

During the 6th hour, the blastoderm nuclei enlarge (Text-figs. 3E; 4B; Plate 1,fig. D) becoming oval, with the long axis radially aligned. At the same time theboundary between blastoderm and yolk becomes regular and distinct, evidentlyas the incorporation of cytoplasm from the interior into the blastoderm is com-pleted. The vitellophages all migrate towards the longitudinal midline of theyolk, while the pole cells divide once more to reach their definitive number of 32(Text-fig. 3G).


The 7th hour of development is marked by the onset of processes transform-ing the syncytial into a cellular blastoderm. The surface convexities alreadyassociated with the blastoderm nuclei indicate the limits of the future cells and

syncytial blastoderm

pole cells

A

secondaryvitellophagenulceus

B

cellular blastoderm

yolk sac

Cpole cells

TEXT-FIG. 4. A, sagittal section through posterior end of 5-hour embryo, B, frontal section throughposterior end of 6-hour embryo, c, sagittal section through posterior end of 7^-hour embryo.

during the 7th hour radial cell membranes begin to push down between andbeyond the nuclei, which at the same time develop nucleoli (compare Text-figs.3 E, F). In embryos 7 hours old the membranes have reached deep into the blasto-derm (Text-fig. 3F; Plate 1, fig. E) but completion of the radial and inner


tangential membranes cutting off the cellular blastoderm at the surface of theyolk takes a further 30 minutes (Text-figs. 3G; 7A; Plate 2, fig. A).

The inner tangential membranes of the blastoderm cells lie not at the boundarybetween blastoderm and yolk but slightly peripheral to it. As a result, a thincontinuous layer of nucleus-free cytoplasm remains as a sac around the yolk andvitellophages (Text-fig. 3G). At the posterior pole of the embryo the inner tan-gential boundary of the blastoderm meets the space beneath the pole cells,which thus lie within a circular polar aperture in the blastoderm. The nucleatedcytoplasmic cone beneath the pole cells is incorporated into the perilecithalcytoplasmic sac (Text-fig. 4c).

Gastrulation

As soon as the cellular blastoderm is complete a series of migrations anddivisions commences among its cells, and those which will give rise to the internalorgans of the larva move into the interior. At the same time various parts of theembryo acquire characteristics which allow them to be histologically identifiedas organ primordia. This complex of processes can be placed under the heading

pole cells—-"~g«- J ___proctodaeum T ^ V /

^-—^—~v^^posterior \ ^ ^ ^ Omid-gut ^^"^><^>*^

extra - embryonicectoderm >

/embryonic

ectoderm

/trochealrudiment

. cerebralqanqlion

stomodaeum\anteriormid-gut

mesodermmesoderm marqmal salivary cephalic ventral

strip qland furrow nerve cord

TEXT-FIG. 5. Presumptive areas of blastoderm in D. tryoni, lateral view.

of gastrulation. Its description is facilitated by preliminary reference to a pre-sumptive area map of the blastoderm, derived not from differences detectablein the blastoderm by the methods employed in this study but by extrapolationof subsequent events back on to the blastoderm areas in which they are initiated(Text-fig. 5). It remains for detailed experimentation to decide how far theseareas can be separated in the blastoderm or earlier in terms of ooplasmicsegregation or to what extent their establishment depends on causal processesof epigenesis during cleavage and gastrulation, although the evidence availablefor other cyclorrhaphous Diptera suggests that the presumptive areas are deter-mined at a very early stage in development, perhaps even before fertilization.Acknowledging this limitation, however, the apparent presumptive areas shownin Text-fig. 5 are of considerable use in the present task of describing the celllineage of the species.


The main areas which can be discerned are:

1. Mid-ventrally, as a band some 16 cells wide along the length of the blasto-derm, stopping short of either end—the presumptive mesoderm.

2. Immediately lateral to the presumptive mesoderm on either side, a narrowmarginal strip which contributes both to the mesoderm and to the ventralnerve-cord.

3. Antero-ventrally in front of the anterior end of the presumptive mesoderm—the presumptive anterior portion of the larval mid-gut.

4. Surrounding the presumptive anterior mid-gut anteriorly and laterally—the presumptive stomodaeum.

5. Postero-ventrally behind the posterior end of the presumptive mesodermand in front of the pole cells—the presumptive posterior portion of thelarval mid-gut.

6. At the posterior end, distinct from the blastoderm—the pole cells.7. Surrounding the pole cells dorsally and laterally and abutting against the

presumptive posterior mid-gut—the presumptive proctodaeum.8. Occupying the lateral regions of the blastoderm and extending over the

anterior end of the embryo—the presumptive ectoderm of the head andtrunk.

9. Mid-dorsally as a strip stopping short of either end—the presumptiveextra-embryonic ectoderm.

Ignoring for the moment the presumptive imaginal cells, which also finda place within the blastoderm, the events which result in these areas beingseparated out in the spatial arrangement from which organogeny proceeds willnow be considered in turn.

The presumptive mesoderm and marginal strips

As soon as blastoderm formation is complete the mid-ventral band of pre-sumptive mesoderm begins to sink inwards, more rapidly in the midline thanat the sides, while its lateral cells curve towards the midline and take up thespace vacated at the surface by the median cells (Text-fig. 7 A, B ; Plate 2, figs.A, B). Sinking in first takes place in the middle region of the embryo but rapidlyspreads to the ends of the presumptive mesoderm and is obvious over the entireband by 8 hours (Text-fig. 6A). Within the next half hour the lateral borders ofthe gutter so formed approach each other in the midline so that it becomes analmost closed tube (Text-fig. 7, C-F). A connexion is retained, however, withthe cells of the marginal strips. As the presumptive mesoderm sinks in, themarginal cells change from a columnar to a cuboidal form preparatory to con-tributing to the mesoderm itself.

Before mid-ventral closure is completed, i.e. between 8 and 8£ hours, cell-division begins within the mesoderm at a point about one-third of the way alongits length (Text-fig. 7c) and, as closure of the groove proceeds, the divisions


spread both forwards and backwards along the band. The peak of this mitoticactivity is reached between 8 | and 9 hours. The number of cells in the bandrapidly increases and the band itself elongates, growing round the posterior end

pole cells

invoqinofinqmesoderm

procfodoeum

posteriormid-gut

yolk sac

pole cells

mesodermprocrodaeum

extra-embryonicectoderm

embryonicectoderm

stomodaeum

100// extra-embryonicectoderm

anteriormid-gut

pole cells

embryonic ectoderm mesoderm posterior / Proct/ a e u m yolk sac stomodaeum

embryonic'ectoderm

TEXT-FIG. 6. A, diagrammatic sagittal section through 8-hour embryo. B, diagrammatic sagittal sectionthrough 9-hour embryo (compare Plate 2, fig. C). c, diagrammatic sagittal section through 12-hour

embryo (compare Plate 2, fig. F).

of the yolk and forwards along its mid-dorsal surface to the boundary of theanterior third of the embryo (Text-fig. 6 B, c; Plate 2, fig. F). Elongation of themesoderm in this way is part of a complex process of elongation involvingalmost all the presumptive areas of the embryo. Other aspects of the processare described below.


vitellophagesyolk sac procfodaeum

yolkcells

blastoderm posteriormid-gut

mvaqinatmqmesoderm

invaqmatinqmesoderm

mesoderm


ventralgroove

pole cells

posteriormid-gut

embryonicectoderm

mesoderm

embryonic"ectoderm

mesoderm

\mesoderm

marqinalVrip

TEXT-FIG. 7. A, transverse section through embryo fixed 7 hours 40 minutes after oviposition (com-pare Plate 2, fig. A), B, sagittal section through posterior end of 8-hour embryo, c, transverse sectionthrough 8^-hour embryo at level indicated on fig. 1, c. D, transverse section through 8£-hour embryoat level indicated on fig. 1, c. E, transverse section through 8^-hour embryo at level indicated on

fig. 1, c. F, transverse section through 8£-hour embryo at level indicated on fig. 1, c.


While the cells of the presumptive mesoderm are thus dividing, further cellsare added to the band through cell division in the marginal strips (Text-fig. 8A).Finally, towards 9 hours, the edges of the mesodermal band come together,closing and obliterating the mid-ventral groove (Text-fig. 8 A, B) and leaving theband as an irregular multi-layered strip of polygonal cells, triangular in cross-section, extending from a point not far short of the anterior end of the embryoalong the ventral midline, round the posterior end and forward alongthe dorsal surface. At the anterior and posterior ends of the closing groove themarginal cells give rise to a surface epithelium which loses connexion with themesoderm, but over most of its length closure of the groove results in formationby the marginal cells of a middle strand, some 3-4 cells wide, whose cells projectinwards from narrow bases and enlarge at their inner ends so as to retainapparent continuity with the mesoderm.

After 9 hours, mitotic activity in the mesoderm decreases. Once the definitiveband length is achieved at 11 hours, the mesoderm cells spread laterally beneaththe embryonic ectoderm (see below) to form a single layer, completed by 12 hoursafter oviposition. Cell-division within the mesoderm is now infrequent. Duringthe same period (9-12 hours), the mesoderm grows forward laterally on eitherside of the developing anterior mid-gut (see below) almost to the anterior endof the embryo.

The presumptive anterior mid-gut and stomodaeum

The presumptive anterior mid-gut first becomes histologically distinguishablebetween 8 J and 9 hours when its cells, by amoeboid immigration, form the wallsof a pear-shaped depression in front of the anterior end of the mid-ventralgroove of the immigrating mesoderm. By 9 hours after oviposition cells havebegun to be budded off from the apex and posterior surface of this depression(Text-figs. 6B; 8D; Plate 2, fig. C) and later, by 11 hours, also from its side walls,to form a mass of actively dividing small cells immediately internal to the depres-sion itself. As the number of cells increases the mass grows posteriorly in theventral midline so that by 12 hours it forms a wedge between the surface of theyolk and the mesodermal band (Text-figs. 6c; 8E; Plate 2, fig. F). At the sametime, as described above, the anterior end of the mesodermal band grows for-ward on either side of the wedge. While these changes are taking place, closureof the mid-ventral groove immediately behind the anterior mid-gut rudimenttransforms the horseshoe-shaped presumptive stomodaeum into a closed ringat the periphery of the anterior mid-gut depression (Text-fig. 8E).

The presumptive posterior mid-gut, pole cells, and presumptive proctodaeum

The sequence of events through which the posterior mid-gut and proctodaeumcomplete their early development is more complex than the correspondingsequence for the anterior mid-gut and stomodaeum. The pole cells are closelyassociated with them, while elongation of the mesodermal band and its covering


ectoderm (see below) also cause the movement of the posterior rudiments fromtheir initial position to a final dorsal position at the border of the anterior thirdof the embryo. The movement begins about 8 hours after oviposition (Text-figs.6A; 7B), proceeds rapidly as cell-division in the mesoderm reaches its peakbetween 8J and 9 hours (Text-fig. 6B), continues at the same rate during thenext hour, and then slows to completion by 11 hours. It can be followed inliving de-chorionated eggs and a description of events at the egg surface formsa useful preliminary to a detailed description of development. As forward move-ment of the posterior complex begins, a shallow depression, circular in outline,with its posterior border drawn out to a point confluent with the mid-ventralmesodermal groove, forms around the pole cells. As the complex moves forwardalong the dorsal surface the depression deepens, swallowing the pole cells (Text-fig, lc), and by 10 hours, when the forward movement is almost complete, therim is circular anteriorly and has a raised posterior transverse lip. During thelast hour of its forward movement the aperture of the depression closes to atransverse slit with posteriorly curved lateral corners and a slightly raisedanterior lip (Text-fig. ID).

The pole-cell group lies at first with its base in a cone-shaped cavity whosewalls are blastoderm cells and whose floor is the surface of the yolk (Text-fig. 4c).The depression which forms around the pole cells results from elongation andinward migration of the blastoderm cells and initially no distinction can bemade between any of these cells. Those immediately adjacent to the pole cellspenetrate the most rapidly, so that the depth of the hollow cone in which thepole cells he increases and the pole cells sink inwards and are removed from thesurface (Text-fig. 7B). By 8£ hours, however, the immigrating blastoderm cellsimmediately posterior and postero-lateral to the pole cells show a much greaterpenetration than their fellows (Text-fig. 7E). They are quickly displaced fromthe surface to the interior, the cells lateral to them closing over the surface toreplace them. In this way the posterior mid-gut rudiment becomes histologicallydistinct as a mass of elongated cells posterior to the pole cells at the free innerend of the immigrating posterior complex. At the same time the horseshoe-shaped presumptive proctodaeum is transformed into a ring encircling theopening of the depression in which the pole cells lie (Plate 2, fig. D). Completionof the ring is accompanied by closure of the posterior end of the mid-ventralmesodermal groove as described above.

The cells of the proctodaeal ring continue to migrate inwards during the nexthalf hour, at the same time turning through 90° to become radially arranged asthe wall of a proctodaeal tube, those which initially lay closest to the pole cellsforming the inner end of the tube (Text-figs. 6B; 8C; Plate 2, figs. C, D). Theanterior and lateral walls of the tube are established more quickly than theposterior, the formation of which is delayed until the posterior mid-gut cellshave become internal.

The pole cells, sinking in more and more as the proctodaeum opens the way



mesoderm

embryonicectoderm

proctodaeum

marginal strip

embryonicectoderm


procrodaeumembryonicectoderm extra-embryonic ectodern

sfomodaeum

.ectodermof head

neuroblast embryonicectoderm

sromodaeum

TEXT-FIG. 8. A, transverse section through 9-hour embryo at level indicated on fig. 6, B. B, transversesection through 9-hour embryo at level indicated on fig. 6, B. C, sagittal section through posteriormid-gut, pole cell, proctodaeum complex of 9-hour embryo, D, sagittal section through anterior end

of 9-hour embryo, E, sagittal section through anterior end of 12-hour embryo.


for them, remain a distinct mass occluding the end of the tube in contact withthe yolk, with the posterior mid-gut rudiment lying behind them (Text-figs. 6B;8c; Plate 2, fig. D). The proctodaeal tube now begins to increase in length, about9 hours after oviposition, through division of the cells of its walls, while theposterior mid-gut rudiment begins to bud off a mass of small cells between theyolk and the posterior region of the mesodermal band. A few pole cells appearto disintegrate at this stage, since a small amount of granular material is alwaysfound in the proctodaeal lumen vacated by them, but the majority of the cellsremain intact within the pole-cell mass. The condition described above persistsuntil the end of gastrulation (Text-figs. 6c; 8E; Plate 2, fig. F), by which timethe proctodaeum has reached its most anterior position. As a consequence ofthe simultaneous elongation and forward migration of the proctodaeum, its freeinner end, together with the pole cells and posterior mid-gut rudiment, is dis-placed backwards through being dragged against the yolk.

The presumptive embryonic and extra-embryonic ectoderm

By the end of gastrulation, the presumptive embryonic and extra-embryonicectoderm have become converted into a surface layer enclosing the yolk and thevarious rudiments now internal.

The major part of the presumptive embryonic ectoderm forms two broadlateral bands of blastoderm cells and as mid-ventral immigration of the pre-sumptive mesoderm proceeds these bands move ventrally as a whole (Text-figs.7D; 8A). At about 8 hours, as the now internal mesodermal band begins toelongate, the posterior two-thirds of each band of ectoderm becomes narrowerand presses down towards the ventral midline. As a result, the more posteriorcells of the ectodermal bands move over the posterior pole of the embryo andforwards along its dorsal surface (Text-fig. 8 A, B; Plate 2, fig. C). Little mitoticactivity is evident in the ectoderm during this time. The two bands, separated inthe midline by the middle strand derived from the marginal strips (see above)thus join the elongating mesodermal band in pushing the posterior mid-gut-polecell-proctodaeum complex into its antero-dorsal position. The anterior third ofeach ectodermal band is much wider than the posterior two-thirds, reachingalmost to the dorsal surface, and does not appear to be involved in the processof elongation.

Elongation of the embryonic ectoderm is completed by 11 hours. Before thistime, separation of rudiments within the bands begins, so that no clear distinc-tion can be made between the end of gastrulation and the beginning of organo-geny. In two ectodermal regions, on either side of the middle strand behind theanterior third of the embryo (Text-fig. 9E) and latero-dorsally on either side ofthe anterior third of the embryo, some of the original blastoderm cells showcharacteristic nuclear changes (enlargement of the nucleus, entry into prophaseof mitosis) and migrate from the surface to the interior to lie between the surfacecells and the cells of the mesodermal band. Here they begin to divide. These

5584.10 S


cells, as will subsequently be shown, are the neuroblasts from which the gangliaof the central nervous system arise. They form a row some 8 cells wide on eitherside of the ventral midline and a large patch on either side antero-dorsally. Thecells which remain at the surface in their vicinity undergo some tangentialdivisions, maintaining a continuous surface layer without decrease in area.

The presumptive embryonic ectoderm which covers the anterior end of theembryo and borders the presumptive stomodaeum undergoes almost no changeduring gastrulation, remaining as a simple columnar epithelium (Text-fig. 8E).Towards 12 hours, however, scattered mitoses are seen in it, a preliminary tosubsequent changes contributing to the formation of the head.

As the presumptive mesoderm begins to sink in mid-ventrally and the lateralembryonic ectoderm moves ventrally to replace it, the mid-dorsal blastodermcells change from a columnar to a cuboidal form and the area of surface theyoccupy increases. The extra-embryonic ectoderm thus revealed has a clearboundary with the embryonic ectoderm and can be seen over the posteriortwo-thirds of the dorsal surface before forward migration of the posterior com-plex begins (Text-fig. 6A). While the embryonic ectoderm continues to movetowards the ventral midline, the extra-embryonic ectoderm spreads laterally toreplace it both through attenuation of its cells (Text-fig. 8A) and through cell-division. Then, as the posterior complex is pushed forwards along the dorsalsurface, the extra-embryonic ectoderm is displaced from the dorsal midline(Text-fig. 6 B, c) and drawn back as two dorso-lateral horns which finally reachalmost to the posterior end, separating the dorsal and ventral portions of theectodermal bands (Text-fig. ID). Only at its anterior margin does the broadmid-dorsal area of extra-embryonic ectoderm persist in front of the proctodaealanterior lip as a narrow transverse strip joining the two areas.

Over the anterior third of the embryo the dorsal blastoderm cells behavedifferently. The embryonic ectoderm of this region is broad, as described above,and shows little ventral displacement as the presumptive mesoderm sinks in.In association with this, no broad area of thinly spread epithelium is formed onthe dorsal surface in this vicinity and only a narrow mid-dorsal strip of blasto-derm becomes cuboidal and reveals itself as extra-embryonic ectoderm. Thestrip is continuous with the broader area posterior to it but develops later, atabout 8 | hours, and is not involved in the displacement of extra-embryonicectoderm caused by forward migration of the posterior complex. It shows a smallamount of cell-division during the early stages of its formation.

The close association between the activities of the embryonic and extra-embryonic ectoderm during gastrulation is further reflected in the occurrenceof cell-<iivisions in the embryonic ectoderm adjacent to the immigrating meso-derm and marginal strips over the anterior third and at the posterior end of eachectodermal band. It is precisely these regions which lack an attenuation andspread of the dorsal blastoderm as extra-embryonic ectoderm compensating forthe loss of the mid-ventral cells from the surface. Such compensation is therefore


gained through cell-division in the embryonic ectoderm adjacent to the meso-derm. The same process is seen at the lateral borders of the posterior mid-gut-pole cell-proctodaeum complex when this begins to sink inwards (Text-fig. 7E).Anterior to the complex, compensatory surface coverage is gained from extra-embryonic ectoderm formation; posteriorly, immigration of mesoderm is itselftaking place.

Temporary surface furrows

It has been shown above that during gastrulation a distinction can be madebetween events taking place in the anterior third of the embryo and those in theposterior two-thirds. The former shows relatively little movement of cells overits surface while the latter shows the vigorous movements of ectoderm andmesoderm elongation and accompanying dorsal displacement of the posteriorcomplex. Certain accessory processes, such as the relative growth of the extra-embryonic ectoderm and the occurrence of cell-divisions in the ventral regionsof the embryonic ectoderm during mesodermal immigration, confirm this distinc-tion. It is therefore significant that at an early stage in gastrulation, as elongationof the embryonic ectoderm and mesoderm begins, a conspicuous fold appears inthe surface of the embryo at the border between the regions whose subsequentbehaviour differs. This fold, the cephalic furrow (Text-fig, lc), is a deep intuck-ing of the blastoderm into the yolk, first appearing laterally at 8 hours andrapidly extending dorsally and ventrally to encircle the embryo almost trans-versely, with a slight ventro-dorsal backward slope. That furrow formation isnot a consequence of elongation is seen from the facts that the two events beginsimultaneously and that the furrow is complete by 8 | hours, when elongationhas hardly begun. The furrow persists through the early period of rapid elonga-tion (Text-fig, l c ; Plate 2, figs. D, E), cell-division in the mesoderm beginningat the level of the furrow and spreading from here both forwards and backwards,but disappears between 9£ and 10 hours so that the final stages of elongation arecompleted without it (Text-fig. ID). Subsequent events reveal that the cephalicfurrow lies approximately at the boundary between the future maxillary andlabial segments of the head.

Behind the cephalic furrow a similar but less-conspicuous furrow develops,persisting through the same period of development (Text-fig, lc). This secondfurrow does not cross the dorsal surface of the embryo. As elongation of theembryonic ectoderm and mesoderm proceeds, four further pairs of temporarylateral furrows form (Text-fig, lc), but these are shallower and more short-lived than the first (cephalic) and second furrows, being evident only between8£ and 9 | hours, i.e. during the first phase of rapid elongation.

The yolk

As already described, the yolk at the end of blastoderm formation is enclosedby a cytoplasmic sac, anucleate except at the extreme posterior end, while a row


of vitellophages occupies the longitudinal midline of the yolk mass (Text-fig. 3G).During gastrulation, the immigration of cells into the interior of the embryodisplaces the yolk mass in a number of places (Text-fig. 6 A, B, C), but none ofthe cells enters the yolk itself. Formation of the cephalic furrow is also associated,perhaps causally, with a corresponding furrowing of the yolk. The vitellophages,on the other hand, move away from the midline, some becoming scatteredthrough the yolk while the remainder migrate to the yolk surface and fuse withthe perilecithal cytoplasmic sac, transforming it into a thin, nucleated epithelialyolk sac (Text-figs. 6 A, B, C; 8E).

Organogeny of the larval organs

No attempt is made below to present a full account of segmentation or todescribe in detail the morphogenesis and histogenesis of the muscular system,tracheal system, peripheral nervous system, and sense organs. In general theirdevelopment follows the course described by Poulson (1950) for Drosophilamelanogaster. Particular attention is paid, however, to the development of thegut, Malpighian tubules and gonads, and to the fate of the extra-embryonicectoderm.

The general course of development after gastrulation

As organogeny proceeds, the embryo undergoes a number of general changestypical of cyclorrhaphous Diptera.

Three hours after the end of gastrulation, the grooves which delineate thethree post oral head segments, mandibular, maxillary, and labial, appear on thesurface ventro-laterally on either side. After a further 3 hours, the limitingannuli of the trunk segments begin to form in antero-posterior succession aspaired ventro-lateral transverse grooves, the 3 thoracic and 8 abdominal seg-ments being delineated by the end of the 24 hours. At the same time, shorteningof the germ-band occurs. The trunk segments concentrate on to the ventralsurface, the proctodaeal opening is drawn back to its definitive position at theposterior end of the trunk, and the extra-embryonic ectoderm is redistributedover the dorsal surface. Within the next 4 hours dorsal closure is completed, theextra-embryonic ectoderm is resorbed, and the inter-segmental grooves extenddorsally and ventrally to encircle the body.

During shortening of the germ-band, involution of the head begins. The headlobe is pushed antero-dorsally by the crowding forward of the mouthpart seg-ments and both head lobe and mouthpart segments are then infolded within thethoracic segments. Involution is completed in approximately 12 hours. Muscularactivity soon follows, waves of contraction passing forwards along the trunkwhile the mouth hooks are repeatedly protruded and withdrawn. As develop-ment nears completion, activity increases, ultimately resulting in rupture of thevitellrne membrane and chorion by the mouth hooks and escape of the larvathrough the tear.


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TEXT-FIG. 9. A, transverse section through anterior end of 14-hour embryo. B, transverse sectionthrough 14-hour embryo at level of posterior mid-gut rudiment, c, transverse section through 16-hourembryo at level of proctodaeum. D, transverse section through 16-hour embryo at level of posterior

mid-gut rudiment, E, transverse section through 18-hour embryo at level of salivary glands.


Accompanying shortening of the germ-band, dorsal closure and involutionof the head, the organ primordia separated out through gastrulation continuetheir development. The establishment of each major organ system from itsprimordia is described below.

The gut

Disregarding the complex cephalopharyngeal apparatus formed as a result ofinvolution of the head, the gut of the newly hatched larva comprises a tubeof varying diameter, with a lining epithelium covered externally by a thin coatof circular muscle. It can be divided into:

(a) The oesophagus, a thin tube with a cuticular lining, flattened epitheliumand pronounced circular muscle coat, extending from the posterior endof the pharynx through the circum-oesophageal nerve ring.

(b) The proventriculus, into which the oesophagus continues as a muscularcore, surrounded by a layer of pale-staining cells and an outer coat ofcuboidal epithelium staining darkly with haematoxylin.

(c) The mid-gut, a convoluted tube whose epithelial wall is continuousanteriorly with the outer coat of the proventriculus. The mid-gut epi-thelium is cuboidal throughout.

(d) The rectum, beginning antero-laterally at the point at which the Mal-pighian tubules arise from the gut and looping forwards and upwards torun back in the dorsal midline above the coils of the mid-gut before turn-ing down to the anus. The rectal epithelium is irregular and pale stainingand lined by a thin cuticle.

The development of this elaborate tube from its four initial rudiments, thestomodaeum, proctodaeum, and anterior and posterior mid-gut, takes place asfollows.

The stomodaeum (Text-figs. 9 A, E; 10) increases in length through cell-division in its epithelial wall, growing horizontally back beneath the yolk sacand then pushing up into it so that the surface of the yolk sac is infoldedanteriorly. By the time shortening of the germ-band is complete, the stomodaeumhas reached its definitive length, extending from the mouth to the first thoracicsegment as a narrow tube with a cuboidal epithelial wall. As it grows thestomodaeum buds off two small dorsal diverticula, one behind the other. Thefate of these has yet to be followed, but they resemble in origin the rudiments ofthe stomatogastric ganglia of Drosophila melanogaster (Poulson, 1950) andCalliphora erythrocephala (Ludwig, 1949).

The stomodaeum now differentiates directly into the oesophagus and thecentral core and intermediate cell layer of the proventriculus. With involutionof the head, the embryonic mouth is carried inwards and the oesophagusdisplaced posteriorly to lie in the first and second abdominal segments.

As the stomodaeum begins to grow it pushes the enlarging wedge of anterior


mid-gut cells before it (Text-fig. 9A), eventually as far as the first thoracic seg-ment. At the same time, beginning at 14 hours, the wedge itself proliferates twolateral arms which embrace the yolk sac and grow latero-ventrally along it,reaching the posterior end of the embryo in approximately 3 hours (Text-figs. 9 c, D ; 10). A second proliferation of cells occurs on the dorsal surface ofthe wedge between 14 and 15 hours, the products in this case penetrating intothe yolk sac as tertiary vitellophages.

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TEXT-FIG. 10. Diagrammatic sagittal section through 18-hour embryo.

The proctodaeum (Text-figs. 9c; 10) also continues to grow through cell-division in its epithelial wall, pushing before it both the posterior mid-gut rudi-ment and the mass of pole cells. By 14 hours the pole cells can be separated intotwo distinct histological types. Sixteen of the cells show enlargement of thenucleus together with a cytoplasmic change which causes them to stain pinkish-brown with the stains employed, in contrast to the bluish-pink coloration of allother cells. The 16 cells then migrate out of the pole-cell mass to lie scatteredbeneath the enlarging posterior mid-gut rudiment (Text-fig. 9B). Their furtherfate as definitive germ-cells is described below. The remaining pole cells multiplyand within a further 2 hours come to form the ventral wall of the distal end ofthe proctodaeum. Meanwhile the proctodaeum continues to grow and at 16hours produces two pairs of diverticula, one pair laterally placed (Text-fig. 9c),the other dorsally placed immediately behind the first pair at the distal end ofthe tube. The further development of these, the rudiments of the Malpighiantubules, is described below.

Subsequent development of the proctodaeum is complicated by the onset ofshortening of the germ-band during the 18th hour (Text-fig. 10). As the procto-daeal opening is drawn posteriorly, the elongating proctodaeum folds on itselfto give a proximal arm running anteriorly beneath the extra-embryonic ectodermand a distal arm running posteriorly beneath the proximal arm. This arrange-ment persists throughout shortening, the proximal arm growing more rapidlythan the distal so that the inner end of the proctodaeum finally lies anterior tothe anus. Once shortening is completed at 24 hours, the proximal arm continues


its growth in the dorsal midline, pulling the distal arm forward to lie eventuallyin the 3rd and 4th abdominal segments. At the same time histodifferentiationof proctodaeal into rectal epithelium occurs and the epithelium finally secretesthe lining cuticle of the rectum.

The posterior mid-gut rudiment (Text-fig. 9 B, D), occluding the distal end ofthe proctodaeum, is pushed back along the dorsal surface of the yolk sac, firstas a result of proctodaeal growth, later as a combined result of proctodaealgrowth and displacement due to shortening. At about 15 hours it begins toproliferate two lateral, posteriorly directed arms which grow dorso-laterallyover the yolk sac and within 3 hours reach the posterior end and turn ventrallyto meet the paired anterior mid-gut strands described above (Text-fig. 10).

The posterior ends of the paired mid-gut strands are carried posteriorly asa result of shortening so that by 24 hours the two strands stretch antero-posteriorly along the yolk sac. Within the next 4 hours, as dorsal closure is com-pleted, the cells of the strands spread dorsally and ventrally over the yolk sac toenclose it in an ovoid epithelial sac stretching from the end of the stomodaeumto the end of the proctodaeum. Digestion of the yolk within the sac and of theyolk-sac wall and remaining vitellophages is accompanied by elongation andconvolution of the sac itself and histodifferentiation of its wall as mid-gutepithelium. At the anterior end the cells of the mid-gut overgrow the end of thestomodaeum to form the outer epithelium of the proventriculus, while imme-diately behind the proventriculus the mid-gut epithelium is produced as fourdiverticula which give rise to the mid-gut caeca.

The Malpighian tubulesIn the newly hatched larva two pairs of Malpighian tubules leave the gut at

the junction of the mid-gut and rectum, one pair dorso-laterally and one ventro-laterally. The dorso-lateral parr coil posteriorly through the haemocoele, theventro-lateral parr anteriorly. Each tube has a cuboidal epithelial wall and asmall cylindrical lumen. Both pairs of tubules arise at 16 hours as diverticulaof the distal end of the proctodaeum (see p. 269). As the gut develops, the basesof the tubules are carried with it to their definitive position in the 4th abdominalsegment and at the same time each tubule grows through cell-division beforeundergoing histodifferentiation.

The salivary glandsThe salivary glands originate as two ventro-lateral plates of cells which first

become distinct within the ectoderm of the labial segment at about 16 hours.Each plate invaginates and grows inwards as a blind-ending tube retaininga small surface aperture (Text-figs. 9E; 10). Growing first towards the dorsalsurface, the inner end of each tube turns posteriorly to extend parallel to thestomodaeum through the thoracic segments. By 21 hours the vertical portion


has differentiated as a thin duct with a flattened epithelium, the horizontal por-tion with its cuboidal epithelium remaining widely open. As involution of thehead proceeds, the two ducts are drawn out and at the same time come togetherin the midline at the surface to gain a common opening. The inturning of thelabial segment as part of the cephalopharyngeal apparatus brings this openinginto the ventral wall of the latter, removing it from the body surface. The lumenof each gland becomes filled with secretion before hatching takes place. Thesalivary glands in Drosophila melanogaster develop in an identical manner(Sonnenblick, 1939, 1940, 1950; Poulson, 1950).

The derivatives of the mesoderm

The development of the mesodermal bands after gastrulation has not beenfollowed in detail, but a number of observations have been made. Significantamong them are :

(a) the confirmation of separation of somatic and splanchnic mesodermduring the early stages of completion of the gut;

(b) the lack of any evidence of the development of segmentation in the meso-derm earlier than the final differentiation of segmental muscles of thebody-wall. Segmentation of the embryo of D. tryoni appears to be con-fined to the embryonic ectoderm behind the mouth and all trace of somiteswithin the developing mesoderm is lost.

The splanchnic mesoderm arises from cells of the anterior and posterior endsof the mesodermal bands which congregate around the stomodaeum and procto-daeum respectively during the first 6 hours of gastrulation (Text-fig. 10) andfrom two longitudinal strands of mesoderm which separate off from the pairedmesodermal bands between 18 and 24 hours to lie alongside the distal end ofthe stomodaeum and the mid-gut strands (Text-fig. 9B). By the time shorteningis complete the splanchnic mesoderm of the stomodaeum and proctodaeum hasformed a thin epithelial layer over each, the future circular muscle layer. Thenas the mid-gut strands spread over the yolk sac, the splanchnic mesodermstrands spread with them. From the resulting outer layer of cells arises the cir-cular muscle of the mid-gut.

The somatic mesoderm shows little change between 12 and 24 hours otherthan cell multiplication and spread as a mesenchyme particularly in the head(Text-fig. 9, A-E). AS dorsal closure proceeds, however, it begins to histo-differentiate and by the time closure is complete it has given rise to the muscula-ture of the segments and cephalopharyngeal apparatus and to the fat-body,conspicuous in the haemocoele on either side of the trunk as an irregular plateof vacuolated cells extending throughout the abdomen. A small number ofsomatic mesoderm cells also contribute to the developing gonads (see below).The larval heart arises from mesodermal cardioblasts.


The gonadsThe gonads of the newly hatched larva are a pair of ovoid bodies embedded

in the fat-body dorso-laterally in the 5th abdominal segment. Sexes are indistin-guishable, each gonad comprising 8 germ cells with large nuclei and prominentnucleoli, compacted together and covered by a thin epithelium. One or two smallcells lie as interstitial cells among the germ cells.

As has already been pointed out, the 16 germ cells which enter the two gonadsare modified pole cells which at 14 hours lie scattered beneath the developingposterior mid-gut rudiment (Text-fig. 9B). From this position 8 cells migrate oneach side through the growing posterior mid-gut strands to enter the adjacentmesodermal band, so that by 18 hours they are lodged among the mesodermcells of the 6th and 7th abdominal segments (Text-fig. 10). As shortening of thegerm-band takes place, the germ cells are carried within the contracting meso-derm, at the same time moving farther anteriorly and towards each other tocome together at 22 hours as a longitudinal row on each side adjacent to thenow distinct splanchnic mesoderm in the 5th abdominal segment. Within thenext 2 hours each row of germ cells compacts into an oval mass which becomesinvested by a thin coat of somatic mesodermal cells. As dorsal closure and meso-dermal differentiation proceed, the gonads are carried upwards to their definitivedorso-lateral positions adjacent to the developing fat-body. Beyond 28 hoursthey show no further change before hatching.

The central nervous systemIt has already been pointed out that the neuroblasts of the presumptive ner-

vous system begin to separate from the embryonic ectoderm and to histo-differentiate and bud before the end of gastrulation. As this process continues,both the paired cerebral masses and paired longitudinal nerve-cords push deeplyinto the interior, displacing the mesoderm (Text-fig. 9, A-E). Superficial seg-mentation is not reflected in the developing central nervous system, neither partof which is segmentally subdivided. The paired ventral nerve-cords, however,follow the segmental annuli in developing in an antero-posterior sequence. Bythe time shortening of the germ-band begins the central nervous system is welldeveloped and as shortening proceeds, it begins to separate from the surfacelayer of hypodermis and move into the interior. The middle strand, whichremains distinct until this stage (Text-fig. 9E) is drawn in with it and subsequentlyincorporated into the ventral nerve-cord. Between 24 and 28 hours separationof the central nervous system from the hypodermis is completed and at the sametime the two cerebral masses fuse in the midline above the oesophagus and oneither side ventrally with the anterior end of the ventral nerve-cord. Segmenta-tion also becomes apparent in the ventral nerve-cord in segmental fusion of thepaired cords and the appearance within the ganglia of zones of neuropile linkedby paired intersegmental commissures. Once isolated from the hypodermis, the


central nervous system begins to condense, so that by hatching it is concentratedwholly within the first 4 abdominal segments and has lost its segmental pattern.Displacement of the circumoesophageal nerve-ring backwards to the secondabdominal segment is a consequence of involution of the head. Development ofthe nervous system in D. tryoni is typical of Cyclorrhapha.

The trachea! system

Typical of Cyclorrhapha, the larva of D. tryoni has two main longitudinaldorso-lateral tracheal trunks giving off small segmental branches and openingposteriorly by a pair of large postero-dorsal spiracles on the 8th abdominalsegment. Later in larval life a second pair of large spiracles arises at the anteriorends of the tracheal trunks in the prothoracic segment. The origin of the trachealtrunks lies in paired lateral segmental invaginations of the embryonic ecto-derm in the 3 thoracic and first 7 abdominal segments, arising simultaneouslyduring the 16th hour (Text-fig. 9D). Each invagination grows anteriorly andposteriorly to link with those adjacent to it and at the same time breaks its con-nexion with the surface, establishing the paired trunks by the time shorteningof the germ-band is complete. The posterior ends of the invaginations of the7th abdominal segment grow posteriorly on either side of the proctodaeum tomake contact with the developing hypodermis on either side of the anus, inwhich position the two definitive spiracles break through following dorsalclosure.

The hypodermis

The major part of the hypodermis develops directly out of the presumptiveembryonic ectoderm, which undergoes little change other than scattered cell-divisions until dorsal closure begins, but then shows cell-division and transforma-tion from a columnar to a cuboidal epithelium as it spreads over the dorsalsurface. Mid-ventrally and antero-dorsally on the head, hypodermal cells per-sist and spread at the surface as the central nervous system moves into theinterior, so that by 28 hours a complete differentiated hypodermis covers theembryo. The segmental annuli, which first appear at 16 hours and are completedby 28 hours, originate as folds in the developing hypodermis. The special deriva-tives of the embryonic ectoderm of the head lobe and mouthpart segments whichline the cephalopharyngeal apparatus have not yet been studied in detail.

The extra-embryonic ectoderm

Throughout its development, the extra-embryonic ectoderm shows no traceof extension as amnion or serosa. Its distribution at the end of gastrulation isheld until shortening of the germ-band redistributes it as a band along the dorsalsurface once more. With dorsal closure, it is resorbed, disappearing withouttrace by 28 hours.

The timetable of embryonic development in D. tryoni and the cell lineage of

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its larval organs (Text-fig. 11) are summarized below. The discussion whichfollows reviews aspects of dipteran embryology for which the present studyprovides new information.

Timetable of development from oviposition to hatching at 25° C.

Hours0 - | Maturation of egg, fusion of pronuclei, formation of zygote nucleus.| - 1 1st and 2nd synchronous mitoses, 4 cleavage nuclei.1-2 3rd-6th synchronous mitoses, 64 cleavage nuclei.2-3 7th mitosis, invasion of blastoderm by 90 cleavage nuclei, 38 remaining

as vitellophage nuclei; 8th, 9th, and 10th synchronous mitoses, 720 sur-face nuclei, 300 vitellophage nuclei.

3-4 1 lth and 12th synchronous mitoses of surface nuclei, giving 2,880; 4 polecells cut off posteriorly, dividing once to give 8.

4-5 13th synchronous mitosis of surface nuclei, giving definitive number of5,800. Pole cells divide once to give 16.

5-6 Syncytial blastoderm completed, vitellophages migrate to midline, polecells divide once to give definitive number of 32.

6-7-| Syncytial blastoderm becomes cellular, perilecithal cytoplasmic sacformed.

7^-12 Gastrulation, separation of germ-band from extra-embryonic ectoderm,elongation of germ-band, invasion of perilecithal sac by vitellophagesto form nucleated yolk sac.

12-17 Early stages of organogeny, delineation of mouthpart segments.17-24 Shortening of germ-band, delineation of trunk segments, beginning of

involution of the head.24-28 Dorsal closure, resorption of extra-embryonic ectoderm, enclosure of

yolk by mid-gut, continued involution of the head, rapid histodifferentia-tion.

28-32 Completion of involution of the head, continued histodifferentiation,elongation and convolution of mid-gut, beginning of condensation ofthe central nervous system.

32-42 Completion of histodifferentiation, condensation of central nervoussystem, resorption of yolk, onset of muscular activity, hatching.

DISCUSSION

Cleavage and blastoderm formation in DipteraCleavage

In D. tryoni at 25° C , the first 10 synchronous cleavage mitoses are eachcompleted in 15 minutes and the last 3 each in 30 minutes. This rate of cleavageis typical of known cases within the order. In Drosophila melanogaster eachmitotic cycle in cleavage occupies 10 minutes at 25° C. (Huettner, 1933;


Rabinowitz, 1941a; Sonnenblick, 1950) and in other Cyclorrhapha the blasto-derm with its several thousand nuclei is completed within a few hours of ovi-position (Reith, 1925; Auten, 1934; Scott, 1934; Fish, 1947; Hagan, 1951;Breuning, 1957; Imaizumi, 1958). Cleavage rates in the more primitive Nemato-cera have not been closely studied, but in Culex molestus, at 18° C , one mitoticcycle is completed every 20 minutes (Christophers, 1960) and in C. pipiens oneevery 15 minutes for the first 6 and one every 25 minutes for the remaining5 cleavages (Idris, 1959, 1960).

In contrast to the relative constancy of cleavage rates in different species, thenumber of synchronous nuclear divisions taking place during cleavage and thetiming of nuclear invasion of the egg surface are highly variable. In D. tryoni(p. 252), 7 synchronous divisions precede invasion of the surface and 6 follow,giving approximately 5,800 blastoderm nuclei. In Drosophila melanogaster, 8 syn-chronous divisions precede invasion of the surface (Huettner, 1935) and 4 follow(Sonnenblick, 1950), giving 3,200 blastoderm nuclei. No satisfactory data areavailable for other Cyclorrhapha, but similar variations are seen among theNematocera. Christophers (1960) has recently shown that in C. molestus nuclearinvasion of the surface follows the 10th synchronous cleavage mitosis, whenapproximately 720 nuclei move outwards and undergo 3 further synchronouscleavages to give between 5,000 and 6,000 blastoderm nuclei. In C. pipiens, incontrast, invasion of the surface begins after the 7th cleavage and the 120 nucleiwhich move outwards undergo 4 further cleavages to give about 2,000 blasto-derm nuclei (Idris, 1959, 1960). In Sciara coprophila (Du Bois, 1932) and Wacht-liella persicariae (Geyer-Duszinska, 1959) nuclear invasion of the surface againfollows the 7th and in Miastor metraloas (Kahle, 1908) and M. americana(Hegner, 1912) the 6th cleavage, but the final number of blastoderm nuclei isunknown.

Blastoderm formation

The differences in cleavage discussed above are not accompanied by differ-ences in the mode of blastoderm formation. Indeed, blastoderm formation inD. tryoni (p. 255) is identical with that in Drosophila melanogaster (Child &Howland, 1933; Poulson, 1937; Rabinowitz, 1941a; Sonnenblick, 1947, 1950)even in such details as the simultaneous appearance of nucleoli in the blastodermbefore cellularization begins and the cutting off by the inner tangential boundariesof the blastoderm cells of a cytoplasmic sac around the yolk. In other Cyclor-rhapha, blastoderm formation is essentially similar, although the cytoplasmicsac of Dacus and Drosophila has not been described for other species. In Muscadomestica (Reith, 1925), Calliphora vomitoria (Kowalevsky, 1886; Voeltzkow,1888, 1889), C. erythrocephala (Graber, 1889; Noack, 1901; Pauli, 1927), Luciliasericata (Fish, 1947), and Phormia regina (Auten, 1934) the syncytial blastodermshows distinct outer and inner layers, the secondarily incorporated materialof the cytoplasmic reticulum being separated from the initial material of the


cytoplasmic haloes by a thin layer of yolk, but the latter is absorbed during gas-trulation and appears to be of little importance. In Nematocera, little attentionhas been paid to the details of blastoderm formation in any species, but the workof Idris (1960) indicates a close similarity in this respect between Culex pipiensand the Cyclorrhapha.

Pole cells

The cutting off in D. tryoni of pole cells which incorporate the posterior poleplasm and polar granules (p. 254) and lose mitotic synchrony with the cleavagenuclei is a characteristic dipteran developmental feature, but the number andmode of origin of the pole cells varies between species. D. tryoni differs fromother Cyclorrhapha in always producing a fixed number of pole cells, 4, whichare cut off during the 12th cleavage and subsequently divide in synchrony threetimes to give 32. In Drosophila melanogaster (Huettner, 1923; Geigy, 1931;Poulson, 1937; Rabinowitz, 1941a; Sonnenblick, 1950) a variable number ofnuclei, between 3 and 11, enter the posterior pole plasm and are cut off duringthe 8th-9th cleavages in pole cells which multiplyin an irregular manner to give be-tween 36 to 73 cells (Rabinowitz, 1941a). Calliphom erythrocephala (Noack, 1901),C. vomitoria (Kowalevsky, 1886), Luciliasericata(Fish, 1947), and Phormiaregina(Auten, 1934) also initially cut off a variable number of cells and the only knownparallel to the condition in D. tryoni is in Melophagus ovinus (Lassman, 1936),which initially cuts off 12 pole cells. In the Nematocera, pole cell origin is alwaysprecise but there is no constancy in its timing or in the number of cells in differ-ent species. In Culex pipiens (Idris, 1959, 1960) 6 cells are cut off following the7th cleavage and divide once in synchrony to give a group of 12. In Sciara copro-phila (Du Bois, 1932), 2 cells are cut off after the 6th cleavage and divide severaltimes to give a group of 22-28. Two cells are also cut offin Phytophaga destructor,but after the 4th cleavage, then dividing to give a group of 16 (Metcalfe, 1935).In Chironomus a single cell is cut off after the 2nd (Weismann, 1863; Ritter, 1890;Hasper, 1910,1911; Yajima, 1960), and in Miastor after the 3rd cleavage (Kahle,1908; Hegner, 1912, 1914; Nicklas, 1959) and divides three times to give agroup of 8.

Vitellophages

While the blastoderm and pole cells are forming, a number of cleavage nucleiand associate cytoplasmic haloes are always set aside as vitellophages within theyolk. Their mode of origin and final number in D. tryoni, however, is far fromcharacteristic. In this species (p. 253), about 38 of the 128 nuclei resulting fromthe 7th cleavage remain behind in the yolk as the others invade the egg surfaceand undergo 3 (sometimes 4) further mitoses in synchrony with the surface nucleito give approximately 300 (sometimes 600) primary vitellophages. At the sametime, 2-5 of the nuclei which invade the posterior pole of the eggs are left behindin the cytoplasmic sac at the yolk surface when the pole cells are cut off, and of


these, one or two reinvade the yolk with cytoplasmic haloes as secondary vitello-phages. In Drosophila melanogaster, in contrast, nuclear counts made by Sonnen-blick (1950) indicate that approximately 50 of 256 nuclei produced at 8th cleavageremain with their cytoplasmic haloes in the yolk. Since Poulson (1950) puts thedefinitive number of primary vitellophages at about 100, it appears that thenuclei undergo only one synchronous mitosis after separation, though Poulsonagrees with Rabinowitz (1937, 1941a) that they then become polyploid througha single endomitosis. Rabinowitz (1941&) also records reinvasion of the yolkby a number of blastoderm cells as secondary vitellophages, but Poulson wasunable to confirm this. Between 20 and 30 pole cells, however, wander backbetween the blastoderm cells into the yolk (Rabinowitz, 1941 a, b; Sonnenblick,1941, 1950; Poulson, 1947, 1950) and are generally interpreted as secondaryvitellophages (tertiary vitellophages of Rabinowitz), though Poulson (1950,1959) and Poulson & Waterhouse (1960) have suggested an alternative fate forthem (compare p. 284).

In other Cyclorrhapha, primary vitellophages similar in origin to those of Dacusand Drosophila occur in Glossina tachinoides (Hagan, 1951), Lucilia sericata(Fish, 1947), Phormia regina (Auten, 1934), and Melophagus ovinus (Lassman,1936), while in Phormia and Melophagus in addition and in Musca domestica(Reith, 1925), Calliphora vomitoria (Voeltzkow, 1888, 1889), and C. erythro-cephala (Noack, 1901) exclusively, they arise by reinvasion of the yolk byblastoderm cells (sometimes called secondary vitellophages). Secondary vitello-phages like those of Dacus and Drosophila have also been reported in Calliphora,Lucilia, and Melophagus. In Nematocera, little attention has been paid tovitellophage origin. Christophers (1960) describes approximately 550 primaryvitellophages remaining in the yolk in Culex molestus, but in C. pipiens (Idris,1959, 1960) only 32 vitellophages are so formed. In Sciara coprophila (Du Bois,1932) and Miastor metraloas (Kahle, 1908) a small number of vitellophages isformed by reinvasion of the yolk by blastoderm cells, as in Musca, &c.

Thus while the rate of synchronous cleavage and the mode of formation ofthe blastoderm vary little among species, no fixed pattern can be discerned inthe number of cleavage divisions, the timing of nuclear invasion of the egg sur-face and the number and mode of origin of the pole cells and vitellophages.

Gastrulation in Diptera

It has already been shown (p. 256) that the complexities of gastrulation inDiptera are best approached by preliminary reference to the presumptive areasof the cellular blastoderm. The detailed resemblance of the presumptive areamap in D. tryoni to that presented by Poulson (1950) and recently confirmedexperimentally in part by Hathaway & Selman (1961) for Drosophila melano-gaster is suggestive of a constant pattern of blastodermal presumptive areas inDiptera and a concomitant uniformity of gastrulation processes throughout theorder is discernible from a comparison of gastrulation in D. tryoni and other


species. As the following discussion shows, only minor differences are found andmany of these appear to be differences in interpretation of essentially similarprocesses.

Elongation of the germ-band

The concentration in D. tryoni of the embryonic presumptive rudiments withinthe ventral and lateral areas of the blastoderm as a germ-band and the elonga-tion of the germ-band during gastrulation in such a way that its posterior endpushes forward along the dorsal surface of the embryo has been noted in alldescribed cases. A much less extensive simultaneous extension of the anteriorend of the germ-band over the anterior pole occurs in Drosophila melanogaster(Ede & Counce, 1956), Glossinia tachinoides (Hagan, 1951), Melophagus ovinus(Hardenberg, 1929), Sciara coprophila (Butt, 1934), and Simulium pinctipes(Gambrell, 1933) but not in D. tryoni and other species.

Mesoderm

The mid-ventral presumptive mesoderm in D. tryoni behaves in essentiallythe same manner during gastrulation as in Drosophila melanogaster (Sonnen-blick, 1950; Poulson, 1950) and other species, invaginating and subsequentlyspreading as paired ventro-lateral mesodermal bands. In both Dacus and Droso-phila elongation of the invaginated band of mesoderm takes place through rapidcell-division following occlusion of the invagination, but the initiation of mitoticactivity at the level of the cephalic furrow (see below, p. 282) in the formerspecies is not recorded in the latter. The marginal strips which in D. tryoni(p. 260) contribute to the mesoderm before closure of the mid-ventral invagina-tion are also unknown for other species.

Anterior mid-gut

The anterior mid-gut rudiment in D. tryoni (p. 260) is distinct from the meso-derm both histologically and in time, since it begins to invaginate only whenmesodermal invagination is almost complete and then sinks inwards and budsoff a mass of small dividing cells which pushes back between the mesoderm andthe yolk. A similar development of the anterior mid-gut rudiment is describedby Poulson (1950) for Drosophila melanogaster. Other accounts generally failto distinguish early in gastrulation between the mesoderm and the anterior andposterior mid-gut rudiments, but the position of the anterior mid-gut rudimentand the general indications given of its origin suggest a close similarity to Dacusand Drosophila in every case.

Stomodaeum

The early development of the stomodaeum has been little studied in Diptera.Its initial position in D. tryoni as a horseshoe-shaped presumptive area enclosingthe presumptive anterior mid-gut anteriorly and laterally and its subsequent

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closure as a ring at the periphery of the anterior mid-gut invagination arerepeated in Drosophila melanogaster (Poulson, 1950), save that in this speciesthe stomodaeal rudiment closes across the mouth of the anterior mid-gut in-vagination as a stomodaeal plate. In other species the rudiment appears tooccupy the same site, although its position relative to the anterior end of theembryo depends on the extent to which it is carried forwards during elongationof the germ-band. In Melophagus ovinus (Lassman, 1936), for example, it liesantero-dorsally at the end of gastrulation.

Posterior mid-gut, proctodaeum, and pole cells

The development of the posterior mid-gut rudiment must be consideredtogether with that of the proctodaeal rudiment and pole cells with which it isassociated. Events in D. tryoni (p. 260) are particularly clear cut, since theposterior mid-gut can be distinguished from the proctodaeum as soon as thetwo begin to move into the interior and the establishment of the posterior mid-gut rudiment and pole cells at the inner end of the elongating proctodaeal tubeis easily followed. The pole cells, due to their initial position, retain contact withthe yolk surface throughout. In Drosophila melanogaster (Sonnenblick, 1950;Poulson, 1950) there are several points of difference. The presumptive posteriormid-gut in this species occupies the entire area of blastoderm beneath the polecells. During gastrulation it forms a deep invagination into which the pole cellsremaining after separation of the secondary vitellophages (p. 278) sink, separatedfrom the yolk by the posterior mid-gut cells. The proctodaeum is then formedfrom cells at the rim of the invagination as a short tube which pushes theinvagination more deeply into the interior. In earlier accounts (Poulson, 1937;Rabinowitz, 1941a; Sonnenblick, 1941) the initial invagination was also inter-preted as proctodaeal, with posterior mid-gut cells only at its tip, since theMalpighian tubules which in other Diptera have always been assigned a procto-daeal origin (as in D. tryoni, see p. 269) arise in Drosophila from the walls of theinitial invagination. Its interpretation as posterior mid-gut is probably to beaccepted on the grounds that adequate experimental analysis would reveal pre-sumptive Malpighian tubule cells in the blastoderm in slightly different posi-tions relative to the presumptive posterior mid-gut and proctodaeal cells inDrosophila and Dacus.

While available accounts of the development of the posterior complex duringgastrulation in other Diptera, especially that for Melophagus ovinus by Lassman(1936), suggest that events in D. tryoni and Drosophila melanogaster are typicalof Cyclorrhapha, it is difficult in most cases to follow the development of eachseparate rudiment. Re-examination by modern methods, however, would prob-ably reveal in every species a posterior mid-gut rudiment distinguishable earlyin gastrulation and a proctodaeum invaginating as a tube at the rim of theposterior mid-gut and pushing it more deeply into the interior.

In Nematocera, the posterior mid-gut rudiment as an invagination at the


posterior end of the germ-band and the proctodaeum as a secondary invaginationat the same place have been briefly described in Culex molestus (Christophers,1960), C.pipiens(Idris, 1959, 1960), Aedes dorsalis (Telford, 1957), Sciaracopro-phila (Du Bois, 1932; Butt, 1934), Miastor metraloas (Kahle, 1908), and Simu-liumpinctipes (Gambrell, 1933), but a detailed study is wanting. More is known,however, of the behaviour of nematoceran pole cells. Only in C. molestus(Christophers, 1960), C. pipiens (Idris, 1959, 1960), and A. aegypti (Gander,1951) are they known to be carried along the dorsal surface of the embryo atthe end of the germ-band before sinking into the interior. In other species theymigrate inwards before gastrulation begins, either directly into the yolk as inSciara coprophila (DuBois, 1932), or between the cells of the blastoderm into theyolk (Chironomus confinis—Hasper, 1910, 1911; Miastor—Kahle, 1908; Hegner,1912; Phytophaga destructor—Metcalfe, 1935; Wachtliella persicariae—Geyer-Duszinska, 1959; Simulium pinctipes—Gambrell, 1933; and probably Culextarsalis—Rosay, 1959), move through the yolk and separate as two groupswhich come to rest antero-dorsally on either side of the ingrowing posterior mid-gut and proctodaeum. Their further fate is discussed below (p. 285). It isinteresting that the initial polar group of nuclei of the cytoplasmic sac aroundthe yolk in D. tryoni (p. 256) and some of the secondary vitellophages arisingfrom the pole cells in Drosophila melanogaster (p. 278) also move forwardsduring germ-band elongation and come to rest below the posterior mid-gutrudiment. A change of fate is here evidently associated with retention of primi-tive behaviour.

Embryonic and extra-embryonic ectoderm

In every described species of nematoceran and cyclorrhaphan, as in D. tryoni(p. 263) the embryonic ectoderm, which lies lateral and anterior to the invaginat-ing mid-ventral rudiments, remains as columnar blastoderm throughout gas-trulation (save for the histodifferentiation of neuroblasts towards the end of thisprocess, p. 263), while the extra-embryonic ectoderm differentiates by attenua-tion and spread of its cells as a thin dorsal epithelium meeting the embryonicectoderm anteriorly and laterally and the anterior proctodaeal wall posteriorly.As the germ-band elongates, the extra-embryonic ectoderm is displaced in partfrom the dorsal surface and drawn back as two dorso-lateral horns separatingthe ventral (anterior) and dorsal (posterior) portions of the germ-band. Closureof the edges of the mesodermal invagination brings the two lateral bands ofembryonic ectoderm together mid-ventrally and where they meet, a histologi-cally distinct middle strand is established over almost the entire distance betweenthe stomodaeal and proctodaeal rudiments (D. tryoni, see p. 260; Drosophilamelanogaster—Poulson, 1950; Lucilia ceasar—Escherich, 1902 a, b). The cellswhich form the middle strand in D. tryoni are those marginal strip cells whichhave already contributed to the mesoderm before mid-ventral closure (pp. 257 and260). The middle strand is subsequently incorporated into the ventral nerve-cord.


Elongation of the germ-band involves an increase in length of the lateralbands of embryonic ectoderm. The cell activities which bring this about and atthe same time cause, in combination with elongation of the mesoderm, forwarddisplacement of the posterior mid-gut-proctodaeal-pole cell complex, havebeen considered only in D. tryoni (p. 263), Drosophila melanogaster (Sonnen-blick, 1950; Poulson, 1950) and Calliphora erythrocephala (Breuning, 1957). InD. tryoni it is plain that crowding of the presumptive ectoderm cells towards theventral midline behind the cephalic furrow is largely responsible for elongationof the ectoderm, displacement of the posterior rudiments, and redistribution ofthe extra-embryonic ectoderm. Throughout gastrulation, cell-division in theembryonic ectoderm is rare. Only where, as described on p. 264, mid-ventralcrowding is at a minimum, does cell-division play a part in ectodermal spread,i.e. in surface replacement of the immigrating posterior complex and at theedges of the mid-ventral groove in front of the cephalic furrow. A number ofstatements by Sonnenblick and by Poulson indicate that the same processestake place in Drosophila. In particular, cell-division again appears to play aminor role in ectodermal redistribution, being confined, as in D. tryoni, mainlyto the area around the immigrating posterior complex. Breuning (1957) hasattributed an exaggerated importance to this activity, implying that it is a primefactor, together with mesodermal cell-division, in elongation of the posteriorpart of the germ-band. This view is necessitated by her interpretation fromsurface views of elongation of the germ-band in C. erythrocephala throughactivity of a posterior growth zone. Since the posterior region of ectodermalmitotic activity in Drosophila and Dacus is not a growth zone in Breuning's sense,this interpretation must be regarded with caution until more detailed histo-logical studies have been made. It seems more probable that the ectodermalgastrulation movements of Dacus and Drosophila will be found to be typical ofall species and that, as Idris (1959) has recently shown for Culex pipiens,elongation of the germ-band takes place over its entire length behind the cephalicfurrow.

Temporary surface furrows

The temporary furrowing of the surface of the embryo which accompaniesgastrulation and simultaneous elongation of the germ-band in D. tryoni (p. 265)is characteristic of dipteran embryos, but widely different patterns of furrowingoccur in different species and the interpretation of their significance by individualauthors is by no means constant. The cephalic furrow appears to be charac-teristic of Cyclorrhapha and is also present in Culex pipiens (Idris, 1959, 1960)and C. tarsalis (Rosay, 1959). For Drosophila, Poulson regards it as lying at theboundary between head and trunk and functioning as a first step in differentia-tion between the two, but in D. tryoni (p. 266), Calliphora erythrocephala(Breuning, 1957) and Culex pipiens (Idris, 1959, 1960) it lies between the futuremaxillary and labial segments. This more or less arbitrary position, together


with its initial appearance at the onset of germ-band elongation, its effacementwhen the latter is almost complete and the fact that the majority of germ-bandelongation takes place posterior to it, lends support to the view put forwardby Sonnenblick, that it acts as a stabilizing factor against which the forcesinvolved in elongation of the germ-band operate. How the furrow itself forms isnot clear.

It is notable that all other temporary furrows form behind the cephalic furrowduring germ-band elongation and that their arrangement varies widely amongspecies. In D. tryoni, the second transverse furrow behind the cephalic furrowmay assist the action of the former, but the four pairs of more transient ventro-lateral furrows seem to be a simple consequence of buckling during elongation.Similar folds appear in C. pipiens (Idris, 1959, 1960) and C. tarsalis (Rosay,1959). In Drosophila melanogaster such buckling is confined to the extra-embryonic ectoderm in front of the forward-pushing posterior end of the germ-band, several initial folds forming which finally amalgamate as a single deepmid-dorsal fold, soon disappearing. In Calliphora erythrocephala (Breuning,1957) an extreme regularity characterizes the additional temporary folds, whichappear in rapid antero-posterior succession in the extra-embryonic ectodermbehind the cephalic furrow as five transverse folds persisting until elongation ofthe germ-band is almost complete. The earlier workers on Calliphora andLucilia, who described similar additional folds, considered them to be a mechani-cal consequence of germ-band elongation, as in Drosophila and Dacus, butBreuning, on the basis of their regularity and the fact that the cephalic fold isinter-segmental in position, postulates that they represent the inter-segmentalboundaries of the first 5 trunk segments. It has already been shown thatBreuning's assumptions about the mode of growth of the trunk in Calliphora,which are prerequisite to this interpretation of the temporary folds, are un-warranted (p. 282), so that her view of the folds itself must probably be rejected.More needs to be known of the stresses set up by the forces involved in gastrula-tion in Diptera before a satisfactory explanation of surface furrowing can beexpected.

The yolk

The changes which take place in the yolk in D. tryoni during gastrulation,mainly passive changes of shape, are similar in all species. A causal connexionhas been postulated between yolk contraction and cephahc furrow formationin Drosophila melanogaster (Sonnenblick, 1950) but there is no experimentalevidence on this point. The migration of many of the vitellophages to the yolksurface during gastrulation to fuse with the cytoplasmic sac and transform itinto a nucleated yolk sac in D. tryoni (p. 266) is also known for Drosophilamelanogaster (Poulson, 1950) and occurs after gastrulation in Miastor metraloas(Kahle, 1908).


Organogeny in Diptera

The organogeny of the larval organs in Diptera has been subjected to detailedstudy only by Poulson (1950) for Drosophila melanogaster and only aspects ofit for which D. tryoni has provided additional data will be reviewed here. Thegeneral changes, such as external delineation of segments, shortening of thegerm-band, dorsal closure and involution of the head, which accompany organo-geny in D. tryoni in a manner typical of cyclorrhaphous Diptera, have alreadybeen discussed (p. 266).

The gut

The development of the gut in D. tryoni (p. 268) from four initial rudiments,stomodaeum, proctodaeum, and anterior and posterior mid-gut, is typical ofDiptera. The only point of controversy is the contribution made by the polecells in Cyclorrhapha to the wall of the mid-gut. As already stated, they alwayslie at the end of gastrulation in the wall of (D. tryoni) or the lumen of {Droso-phila melanogaster and other species) the posterior mid-gut-proctodaealcomplex. From here, some of the pole cells migrate outwards as definitivegerm-cells (see below, p. 286); some remain associated with the posterior mid-gutrudiment. It was generally assumed by earlier workers that these degenerated,but Poulson (1947, 1950) showed and with Waterhouse (1960), confirmed ex-perimentally for Drosophila melanogaster that they are carried inwards by thetips of the growing posterior mid-gut strands and give rise to the middle region ofthe mid-gut. Poulson & Waterhouse also demonstrated a pole-cell origin of mid-gut cells in Lucilia cuprina and expressed the opinion that in Drosophila all thepole cells entering the embryo via the posterior mid-gut invagination have sucha fate, though it now seems (see below, p. 286) that this is an exaggerated view.In D. tryoni (p. 269) the pole cells remaining in contact with the posterior mid-gut rudiment also contribute to the gut-wall, but without change of position,forming part of the wall of the proctodaeum. Reference to the presumptive fatemaps of Drosophila and Dacus explains both the difference in pole-cell behaviourin the two species and the fact that the pole cells make any contribution to thegut-wall. In the Dacus blastoderm the cells adjacent to the pole cells are mainlyproctodaeal, in Drosophila exclusively posterior mid-gut. Since determinationin cyclorrhaphan eggs takes place very early, possibly before fertilization (Reith,1925; Pauli, 1927; Anderson, 1961; Hathaway & Selman, 1961) and the fate ofthe presumptive areas of the blastoderm is fixed presumably by biochemicaldifferentiation within the periplasm of the egg, it is easy to see that the boundariesof the periplasmic areas might overlap morphologically distinct parts of theblastoderm into which they are subsequently incorporated.

The vitellophages and the yolk sac to which they contribute in Dacus, Droso-phila, and Miastor appear in general to be totally digested with yolk once themid-gut wall is complete, although Poulson (1950) suggests that some of the


cells of the yolk-sac wall in Drosophila melanogaster become incorporated intothe mid-gut wall. There is no evidence of this in D. tryoni (p. 270).

The mesoderm

The contribution of the paired mesodermal bands to the morphology of thelarva in D. tryoni (p. 271) is typical of Diptera, conforming essentially to thepattern described in Drosophila melanogaster by Poulson (1950), but the ques-tion of the segmental composition of the dipteran larva required attention, sinceit has recently been raised by Butt (1960). It is convenient for purposes of discus-sion to consider the trunk separately from the head.

In the developing trunk, segmentation is established first in the ectoderm inantero-posterior succession (e.g. D. tryoni, pp. 266 and 273). Sciara coprophila(Du Bois, 1932; Butt, 1934) and Miastor metraloas (Kahle, 1908) show subsequentsegmentation of the mesodermal bands into paired somites. In Simulium pinc-tipes (Gambrell, 1933), however, paired strands of splanchnic mesoderm separatefrom the mesodermal bands before the somatic mesoderm segments. Drosophilamelanogaster (Poulson, 1950) resembles Simulium in that only the somaticmesoderm segments, while in D. tryoni (p. 271) even the basic segmentation ofthe somatic mesoderm is lost and all segmental derivatives of the mesodermform by direct association with the segmented ectoderm during histodifferentia-tion. There is thus a tendency for the basic mesodermal segmentation in thetrunk of Cyclorrhapha to be lost.

In the head, external segmentation can be recognized with certainty only forthe mouth part segments, which in D. tryoni, as in all species, become externallydelineated before segmentation of the trunk begins. No corresponding segmenta-tion of the mesodermal bands occurs, while in front of the mouth neitherectoderm nor mesoderm retains vestiges of primary segmentation (paired somitesand associate segmental ganglia—Manton, 1949; Anderson 1959) in any specieswhose embryology is known (this account p. 271; Ludwig, 1949; Poulson, 1950;Breuning, 1959; &c). The conclusion of Butt (1960) that the anterior head lobedescribed by Breuning (1957) for the embryo of Calliphora erythrocephala repre-sents the labrum of the dipteran larval head is therefore unfounded, since thepre-oral composition of the head cannot be segmentally analysed.

The gonads

It has been shown for several species that the germ cells of the rudimentarylarval gonads take origin from pole cells while the gonad sheath and interstitialcells take origin from mesoderm. D. tryoni conforms to this pattern (p. 272)and also resembles other Cyclorrhapha in that certain of its pole cells becomeincorporated in the wall of the gut (p. 269). In Nematocera, all the pole cellsbecome germ cells (Ritter, 1890; Kahle, 1908; Hasper, 1910, 1911; Hegner,1912, 1914; Du Bois, 1932; Gambrell, 1933; Metcalfe, 1935; Idris, 1959, 1960).


The explanation of the dual fate of the cyclorrhaphan pole cells in terms of therelative timing of the determination of presumptive areas in the periplasm andformation of the pole cells and blastoderm cells can be extended to explain thedifference in fate between nematoceran and cyclorrhaphan pole cells if accountis taken of recent work by Yajima (1960) on determination in Chironomusdorsalis. In this species determination is not completed until the syncytial blasto-derm stage, whereas the single initial pole cell is cut off after the second syn-chronous cleavage (cf. p. 277). There is thus no possibility that the presumptiveareas of the blastoderm might overlap the pole-cell-forming region. The regularearly formation of pole cells in Nematocera (p. 277) and their strict subsequentdevelopment as germ cells indicates similarly late determination for other mem-bers of the group, although the work of Idris (1959) on Culex pipiens suggeststhat the Culicidae may be closer to the Cyclorrhapha in this respect.

A pole-cell origin of germ cells has been described in Cyclorrhapha for Droso-phila melanogaster, Calliphora erythrocephala, Lucilia cuprina, and Melophagusovinus as well as for the present case of D. tryoni. The migration in the latter of16 pole cells from the posterior mid-gut-pole cell-proctodaeal invagination tothe definitive gonad positions (p. 272) reflects the condition in C. erythrocephala(Noack, 1901) and M. ovinus (Lassman, 1936), although in these two species itis not yet clear how many cells move to the gonads and how many remainbehind, nor what is the fate of the latter. For L. cuprina, evidence of the pole-cell origin of germ cells at present rests with the experimental results of Poulson& Waterhouse (1960) and the actual migration of the cells has not been described.The same series of experiments also forms the basis of assertions by these twoauthors that in Drosophila melanogaster the germ cells arise from pole cellsre-entering the yolk at the posterior pole before gastrulation begins and thatthe pole cells entering via the posterior mid-gut invagination during gastrulationform mid-gut cells only. This contradiction of the classical account of germ-cell origin in D. melanogaster, which describes pole cells migrating to thedefinitive gonad positions after entry via the posterior mid-gut invagination(Huettner, 1940; Sonnenblick, 1941,1950; Aboim, 1945) in the same manner asin D. tryoni, has recently been refuted by Hathaway & Selman (1961) and it cannow be accepted that D. tryoni and Drosophila melanogaster are closely similarand characteristic of Cyclorrhapha in their mode of germ-cell formation.

The extra-embryonic ectoderm

The extra-embryonic ectoderm which differentiates by flattening and attenua-tion of the dorsal cells of the blastoderm and is subsequently displaced in largepart from the dorsal surface by the elongating germ-band, undergoes no furtherchanges in D. tryoni other than return to the dorsal surface when the germ-bandshortens and subsequent resorption when dorsal closure takes place. Suchextreme reduction of the extra-embryonic tissue is typical of Cyclorrhapha,where the only suggestion of amnion formation is in the temporary indrawing


of the edge of the extra-embryonic ectoderm by the invaginating proctodaeumin Drosophila melanogaster (Poulson, 1950). In Nematocera, in contrast, theextra-embryonic ectoderm extends over the germ-band during gastrulation asa typical amnion and serosa. The reasons for the extreme reduction of theembryonic membranes in Cyclorrhapha are obscure and will no doubt remainso until the functions of these membranes in Diptera have been elucidated.

Cell lineage in the Diptera

The general features of cell lineage of the larval organs in Diptera are moreor less constant, as a comparison of the cell-lineage plan presented by Poulson(1950) for Drosophila melanogaster with that given for D. tryoni on p. 274 shows.The major difference between the cell-lineage plan given here for Dacus andthat of Poulson, however, is the introduction of the concept of presumptiveareas. The tentative adoption of this interpretation is a necessary step in formulat-ing hypotheses on which experimentation can be based. If the theoretical conceptof three germ-layers—ectoderm, mesoderm, and endoderm—is retained, thedesign of experiments is limited by the question, what are the differences betweenthe primordia of the three layers? Greater progress will probably be made if thefundamental questions become (a) What are the differences between the pre-sumptive areas of the blastoderm? (b) How are these differences established?(c) What do they lead to in terms of future interaction or autonomous develop-ment?

SUMMARY

1. The egg of D. tryoni undergoes 7 synchronous nuclear cleavages before 90nuclei invade the surface. The surface nuclei then undergo 6 further synchronouscleavages and a blastoderm of 5,800 cells is formed. The 38 nuclei remaining inthe yolk divide synchronously three times to give about 300 primary vitello-phages. At the posterior pole 4 pole cells are cut off, dividing synchronouslythree times to give 32. 2-5 nuclei at the posterior pole form secondary vitello-phages.

2. Gastrulation, involving invagination of anterior and posterior mid-gutrudiments, is accompanied by invagination of mesoderm mid-ventrally and ofpole cells and proctodaeum posteriorly. The dorsal blastoderm thins to extra-embryonic ectoderm, displaced laterally as the germ-band elongates duringgastrulation. Elongation takes place mainly posterior to a temporary cephalicfurrow, behind which a second transverse and four further pairs of temporarylateral folds form. Most of the vitellophages migrate to the yolk surface to forma nucleated yolk sac.

3. Organogeny is accompanied by segmentation, shortening of the germ-band, dorsal closure, and involution of the head. The gut develops from stomo-daeum, proctodaeum, and anterior and posterior mid-gut rudiments. Malpighiantubules arise as outgrowths of the proctodaeum, salivary glands as ventro-


lateral ectodermal plates on the labial segment. The paired mesodermal bandsdo not segment. Splanchnic mesoderm gives rise to visceral musculature, somaticto segmental musculature, fat-body, heart, and gonad sheaths. The primordialgerm cells of the gonads are formed by 16 pole cells. Other pole cells form partof the proctodaeal wall. The central nervous system arises from ventro-lateraland antero-lateral ectodermal neuroblasts separated from the hypodermis, thetracheal system from paired segmental ectodermal invaginations. The remainderof the embryonic ectoderm gives hypodermis; extra-embryonic ectoderm isresorbed at dorsal closure. No amnion or serosa form.

4. Cleavage in D. tryoni is typical of Diptera. The rate of synchronous cleavageand the mode of formation of the blastoderm follow a relatively constantpattern in different species and although the number of cleavage divisions,timing of nuclear invasion of the egg surface and number and mode of originof the pole cells and vitellophages vary, the resulting blastodermal structure andpresumptive areas in the blastoderm are probably constant for the order. Themode of gastrulation is also constant, save for a difference in pole-cell behaviourin Nematocera and Cyclorrhapha. In organogeny, the pole cells of Cyclorrhaphagenerally contribute to the gut-wall, those of Nematocera do not, due to therelative timing of pole-cell formation and determination of the presumptive areasof the blastoderm in the two groups. Gut formation in D. tryoni is otherwisetypical of Diptera, as is the further development of the mesoderm. Segmenta-tion in Diptera is ectodermal, tending to be suppressed in the mesoderm. Pre-oral segmentation is wholly suppressed. The germ cells of dipteran gonadsalways arise from pole cells, the gonad sheaths from mesoderm. In Nematocera,the extra-embryonic ectoderm extends as amnion and serosa; in Cyclorrhaphano embryonic membranes develop. Cell lineage of the larval organs of Dipterais more or less constant among species.

RESUME

Developpement embryonnaire de Dactis tryoni

1. L'oeuf de D. tryoni subit 7 divisions nucleaires synchrones a Tissue des-quelles 90 noyaux colonisent la surface. Ces noyaux de surface subissent alors6 autres divisions synchrones, realisant ainsi un blastoderme de 5.800 cellules.Les 38 noyaux demeures dans le vitellus se divisent 3 fois de facon synchrone,donnant environ 300 vitellophages primaires. Au pole posterieur 4 cellulespolaires se separent, qui se divisent 3 fois de facon synchrone, en donnant32 cellules. De 2 a 5 noyaux forment au pole posterieur les vitellophagessecondaires.

2. La gastrulation, impliquant l'invagination des ebauches anterieure et pos-terieure de l'intestin moyen, s'accompagne de l'invagination du mesodermemedioventralement, et des cellules polaires ainsi que du proctodeum. Le blasto-derme dorsal s'amincit en ectoderme extra-embryonnaire reporte lateralement


lors de l'allongement de la bandelette germinative qui a lieu aii cours de lagastrulation. L'allongement se produit principalement en arriere d'un silloncephalique temporaire. Plus en arriere apparait un autre sillon transverse, puis4 paires de replis lateraux egalement temporaires. La plupart des vitellophagesmigrent a la surface du vitellus pour former un sac vitellin nuclee.

3. L'organogenese s'accompagne de l'apparition des segments, du raccour-cissement de la bandelette germinative, de la fermeture de la region dorsale etde Pinvolution de la tete. Le tube digestif se developpe a partir du stomodeum,du proctodeum, et des ebauches de l'intestin anterieur et del'intestinposterieur.Les tubules de Malpighi apparaissent comme des expansions du proctodeumet les glandes salivaires comme des plaques ectodermiques ventro-laterales dusegment labial. Le mesoderme splanchnique donne la musculature viscerale, etle mesoderme somatique la musculature segmentaire ainsi que le corps adipeux,le cceur et la gaine des gonades. Les cellules germinales primordiales sont repre-sentees par 16 cellules polaires. Le systeme nerveux central derive de neuro-blastes ectodermiques ventro-lateraux et antero-laterauxsepares de l'hypoderme;le systeme tracheal se forme a partir d'invaginations segmentaires paires del'ectoderme. Le reste de l'ectoderme de l'embryon donne 1'hypoderme; l'ecto-derme extra-embryonnaire est resorbe lors de la fermeture dorsale. II ne seforme ni amnios, ni sereuse.

4. La segmentation de l'oeuf chez D. tryoni est caracteristique des Dipteres.Le rythme des divisions synchrones et le mode de formation du blastodermesuivent des modalites constantes. Bien qu'on observe des variations dans lenombre des divisions, dans l'horaire de la colonisation de la surface de l'oeufpar les noyaux, ainsi que dans le nombre et l'origine des cellules polaires et desvitellophages, neanmoins la structure du blastoderme edifie de meme que ladisposition des territoires presomptifs de ce blastoderme demeure vraisem-blablement constant dans tout l'ordre. Les processus de gastrulation sontegalement constant, mise a part une difference dans le comportement descellules polaires entre les Nematoceres et les Cyclorhaphes. Au cours de l'organo-genese, il est de regie chez les Cyclorhaphes que les cellules polaires participenta la paroi du tube digestif, a l'oppose des Nematoceres, en raison de la chrono-logie relative de la formation des cellules polaires et de la determination desterritoires presomptifs dans les 2 genres. L'edification du tube digestif chezD. tryoni est par ailleurs typique des Dipteres, de meme que revolution ulterieuredu mesoderme. Les segments concernent surtout l'ectoderme et tendent a dis-paraitre dans le mesoderme. La segmentation pre-orale est totalement sup-primee. Les cellules germinales des gonades des Dipteres derivent toujours descellules polaires, et le reste de la gonade du mesoderme. Chez les Nematoceres,l'ectoderme extra-embryonnaire s'etend pour former un amnios et une sereuse;chez les Cyclorhaphes, il ne se developpe pas de membranes embryonnaires.La destinee des diverses cellules dans la formation des organes larvaires desDipteres est plus ou moins constante d'une espece a l'autre.


ACKNOWLEDGEMENTS

I would like to thank Dr. M. A. Bateman for the provision of material forthis study, Miss S. McPhail for technical assistance, Mr. L. Congdon for assis-tance in the preparation of photomicrographs, and Dr. S. M. Manton andProfessor D. R. Newth for advice on matters of presentation. The work wassupported by a research grant from the University of Sydney.

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EXPLANATION OF PLATES

PLATE 1

FIG. A. Sagittal section through anterior end of zygote. x 350.FIG. B. Sagittal section through embryo after first cleavage, x 100.FIG. C. Parasagittal section through embryo at 6th cleavage, x 100.FIG. D. Transverse section through middle region of 6-hour embryo, x 210.FIG. E. Transverse section through middle region of 7-hour embryo, x 210.

PLATE 2

FIG. A. Transverse section through middle region of 7-hour 40-minute embryo, x 210.FIG. B. Transverse section through middle region of 8-hour embryo, x 210.FIG. C. Sagittal section through 9-hour embryo, x 100.FIG. D. Frontal section through 9-hour embryo, x 100.FIG. E. Parasagittal section through 9-hour embryo, x 100.FIG. F. Sagittal section through 12-hour embryo, x 100.

{Manuscript received 1: xi: 61)

J. Embryol. exp. Morph. Vol. 10, Part 3

D. T. ANDERSON

Plate 1

J. Embryol. exp. Morph. Vol. 10, Part 3

D. T. ANDERSON

Plate 2

the embryology of dacus tryoni (frogg.) [diptera, trypetidae (= … · the embryology of dacus...

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