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BLASTOKINESIS AND EMBRYOLOGY OF THE PHASMID Clitarchus hookeri

By I.A.N. Stringer*

I N T R O D U C T I O N

New Zealand can perhaps be considered fortunate in having a number of endemic Phasmids, as these stick insects are predominantly a tropical group particularly well known for parthenogenesis, mimicry of plant forms, and colour change in response to their surroundings. However, comparatively little is published about many aspects of the Phasmidae: the only recent detailed papers on their embryology are those of Thomas (1936) who describes the embryology of Carausius morosus (= Dixippus morosus) and Bergerard (1958) who describes that of Clitumnus extradentatus.

The opportunity was therefore taken to study the embryology of Clitarchus hookeri, because it occurs in large accessible populations around Auckland, and the following account is restricted to the embryonic membranes, the movements of the growing embryo or blastokinesis and the development of the externals up to eclosion.

A newly laid egg consists of a large dense mass of yolk spheres and nuclear structures surrounded by a thin periplasm where the cytoplasm is free from yolk. It is somewhat bean shaped and is enclosed by a vitelline membrane and two protecting cuticular envelopes; an inner double thin endochorion which opens at the micropyle and forms the respiratory system, and an outer complex thick exochorion which is calcified.

The axial orientation of the egg is taken from its position in the ovary relative to the axes of the female. Anteriorly the exochorion is produced into a cone shaped operculum (which the nymph pushes off when it hatches) and ventrally into a raised micropylar plate bearing the micropyle.

M A T E R I A L S A N D M E T H O D S

Male and female stick insects were collected once a week from Bethells Beach, 20 miles N.W. of Auckland, and kept in large cages supplied with fresh tea-tree (Leptospermum sp.). The eggs were dropped singly to the floor of the cages where they were collected every morning and kept as daily or weekly batches inside polyethylene foam boxes containing sand wet with 1% nipagin-M-sodium to prevent mould. The inside of the containers, with a temperature usually between 6 4 ° F and 6 5 ° F (but on occasions varying from

*Department of Zoology, University of Auckland. After August, Department of Entomology, University of Alberta.

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6 2 ° F to 6 6 ° F ) , provided favourable conditions for development.

Ten to thirty eggs were removed for study twice each week as it was found that the embryos showed considerable variation in developmental rates; for example, eggs examined one month before hatching might contain fully develop­ed embryos or early embryos. However, most of the eggs hatched 120 to 150 days after laying, although some continued to hatch up to four months after the main eclosion period and produced both male and female nymphs. This problem of varying developmental rates has been encountered by other workers on the Phasmidae.

Owing to the large amount of refractory yolk present the egg could not be sectioned in paraffin by the ordinary methods. Partial success was achieved by using the method of Thomas (1936) of double embedding in celloidin and wax but complete serial sections were only obtained by using Andersons' Dioxan method (1964).

Early embryos had to be fixed (by the Thomas method) before they could be removed from their exochorions. It was not necessary to make whole mounts as the fixative made them sufficiently opaque. Larger embryos could be dissected from fresh eggs. Here the eggs were stuck to a microscope slide in batches of 20 to 30, by a mixture of plastic (from disposable petri dishes) dissolved in chloroform, as this proved better than the commercial solvent type adhesives tried. The slides were then immersed in 4% formalin in Insect Ringers and observed under a binocular microscope. The chorion could then be carefully removed, piece by piece, to examine the embryo in situ, but for most purposes the exochorion could be cracked by gentle pressure from forceps, which were then inserted into the crack so that the embryo 'flowed' out. The very delicate embryo was rapidly hardened by the formaldehyde and could be manipulated within half a minute or so.

E M B R Y O L O G Y

The cleavage nuclei migrate through the yolk to the periplasm on the surface. Most move ventrally (towards the micropyle) and here they multiply into a syncytial mass which spreads around the yolk to form the blastoderm.

Just before the appearance of the embryo a longitudinal area of blastoderm becomes visible ventrally (Fig. 1 A) , although small patches also occur irregularly scattered over the egg. The embryo then develops as a rounded or diamond shaped thickening in this blastodem, just posterior to the micropyle (Fig. I B). It becomes more pronounced until clearly differentiated from the rest of the blastoderm as a flat rounded body with two anterior cephalic lobes (Fig. 1C). At about this time the blastoderm becomes completed dorsally so that the yolk is completely enclosed by it.

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mpl

1mm I I

F I G U R E 1: 1st B L A S T 0 K 1 N E T 1 C M O V E M E N T

Eggs at different stages of embryonic development with their chorions dissected off. A . Egg with lighter region of blastoderm on yolk (dorsal view) B. Embryo developing in blastoderm (dorsal view) C. Embryo with cephalic lobes (dorso-posterior view) D. Embryo beginning first blastokinesis (dorso-posterior view) E. Embryo in mid blastokinesis (dorso-posterior view) F. Embryo after completion of first blastokinetic movement (dorsal view)

mpl., micropyle

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As the embryo begins to elongate it starts its first blastokinetic movement. This consists of a sideways rotation through 180° on the surface of the egg, so that it faces in the opposite direction (Fig. 1 D,E ,F . ) . Segmentation occurs dur­ing blastokinesis and when the embryo has finished turning the segments are completed.

Initially the stomodaeum and proctodaeum show as slight shallow inden­tations between the cephalic lobes and at the posterior end of the embryo respectively. The cephalic lobes show three indistinct serial thickenings on each side. Segments appear serially from the anterior end (behind the cephalic lobes) first. When two abdominal segments can be distinguished the posterior end begins to flex over itself.

The extraembryonic blastoderm, now termed the serosa, overgrows the embryo although it is still attached to the embryo around the edges. Here there

is a region of larger cells which flatten and grow inwards below the serosa to cover the embryo and form the amnion (Fig. 2). By the time segmentation has finished the serosa has lost its connection with the embryo and the amnion completely covers its ventral surface.

Y N

p r

F I G U R E 2:

Longitudinal section through posterior of embryo after completion of 1st blastokinesis.

Am. , amnion; end., endoderm; mes., mesoderm; mes.so., mesodermal somite; pr., proctodaeum; ser., serosa; Y . , yolk; Y . N . , yolk nucleus.

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F I G U R E 3: S E R I E S O F E M B R Y O S A T D I F F E R E N T S T A G E S O F D E V E L O P M E N T

A. Undergoing 1st blastokinesis B. On completion of 1 st blastokinesis C. & D. Before 2nd blastokinesis E. At stage where embryo ruptures through the amnion and serosa

ab., abdominal somite; abp., abdominal appendage; abfx., abdominal flexure; At . , antenna; c , cerci; c l . , cephalic lobe; lb., labrum; mb., mandible; mx., maxilla; th., thoracic appendage.

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Limb buds are formed 3 or 4 segments behind the developing segments as ventral outgrowths or lobes (Fig. 3 A , B , C ) . The first outgrowths are the antennae and the bilobed labrum. As segmentation proceeds the labrum rapidly looses its bilobed form and becomes spatulate to cover the stomodaeum. The mandibles and maxillae develop from the 1st, 3rd and 4th segments behind the antennae, but at no stage could limb buds be distinguished on the 2nd segment. The mandibular and maxillary appendages produce indistinct distal segments (Fig. 3D). Those of the mandibles arc quickly resorbed, but they remain in the maxillae while enlargements grow medially so that they each appear as bunches of three lobes (Fig. 3E).

The thoracic appendages elongate and begin to grow into legs which are directed inwards and posteriorly towards each other so that they eventually touch (Fig. 3C,D,E) .

Eleven abdominal segments develop, although the 1 I th is hard to distinguish from the remaining unsegmented region. However, the 11 th pair of appendages, together with the 2nd, 8th and 9th remain while the rest are resorbed. The second pair enlarge as rounded lobes, the pleuropodia, but the others remain as buds until the end of embryonic life.

The embryo, by this stage, is large and occupies the posterior pole of the egg (Fig. 4 A ) . It is covered ventrally by the amnion and serosa, but just before the second blastokinetic movement it ruptures through these membranes and they retract to enclose only the yolk. Just before rupture the amnion and serosa cannot be distinguished in cut sections, and when they burst the amnion thus joins the serosa to the sides of the embryo and no yolk is lost.

The second blastokinetic movement consists of an enclosing of the yolk by the embryo. An increase in length results from growth of the embryo, but the abdomen continues to grow over itself (so that the posterior end of the flexure stays almost in the same place on the egg) while the head grows anteriorly and the edges grow dorsally (Fig. 4B,C,D) . Thus the yolk eventually completely becomes enclosed within the embryo (Fig. 4E). The abdominal region enlarges proportionately as it grows over the yolk, but the abdomen eventually shrinks in cross-sectional area as the yolk is used up.

The embryo develops during this blastokinesis so that by the time this is finished the embryo resembles the nymph in all important details. The annuli appear to be proliferated from the mid point of the antennae (Fig. 5A) , and the scape and pedicel appear by division of the remaining basal portion. However, the antennal segments are at best only slight constrictions and are hard to follow externally. The mandibles enlarge, while the maxillary palpi begin to show segmentation (Fig. 4 A ) . The second maxillae grow together and fuse to form the labium soon after blastokinesis is completed. The ends of the legs

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1 m m i 1

F I G U R E 4: 2nd B L A S T O K I N E T I C M O V E M E N T

Eggs at different stages of embryonic development; lateral aspects

A. Embryo completed first blastokinetic movement (same as figure 1 F) B. Larger embryo than in A ; enclosed within amnion and serosa C. & D. Stages during dorsal enclosure of yolk E. Completion of blastokinesis F. Fully formed embryo within egg

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A B

ob p

t h 3

F I G U R E 5: SERIES O F E M B R Y O S A T D I F F E R E N T S T A G E S O F D E V E L O P M E N T

A. Head of embryo in B B. Embryo beginning 1st blastokinetic movement; flexure straightened C. Abdomen of embryo in D D. Embryo half completed dorsal closure of yolk

ab., abdominal somite; abp., abdominal appendage; abfx., abdominal flexure; At . , antenna; c c e r c i ; c l . , cephalic lobe; e.,eye; lb., labrum; mb., mandible; mx., maxilla; th., thoracic appendage.

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elongate to grow posteriorly, side by side and then turn outwards away from each other (Fig. 5B,D), to become flexed upon themselves (Fig. 4E) . Primary divisions become apparent and they bend out at the femoral-tibial joint to accommodate their increased length in the limited space. Tarsal segments become apparent in the distal portion of the leg (that is turned outwards). Eyes become apparent in half sized embryos (Fig. 5B) as white spots on lateral enlargements of the head. More are added and as the second blastokinetic movement is completed (Fig. 4D ,E) they begin to darken and eventually consti­tute the individual eye facets. Cerci are formed from the 11th abdominal appendages (Fig. 5C) during the 2nd blastokinetic movement and towards the end of it the 8th and 9th abdominal appendages of the male also disappear while valvifer tubercles develop from the centre of the 8th and 9th limb buds in the female (at this stage they are only swellings).

After completion of blastokinesis the embryo has the appearance shown in figure 4 F . The elongating abdomen moves past the head on the left side of the embryo (no exceptions were noted in over 40 dissections of embryos at this stage).

At the junction of the pro- and mesothorax where the yolk is finally enclosed, there is a pronounced inpushing of the intersegmental region, which develops into an inbending of the thorax (Fig. 4E ,F ) . The three thoracic segments appear distorted and subequal, but they are of equal length when the embryo is dissected out and straightened. The legs lie in a coiled mass in the centre of the egg. The proximal parts of the leg, including the femora, are directed posteriorly, the tibiae are flexed forwards under them, and the tarsi project ventro-posteriorly, outwards, so that the tips project on each side of the abdomen. The femora and tibiae are bowed so that they are parallel to the abdomen and thorax, while the antennae lie on either side, or both to the right, of the abdomen.

The pleuropodia disappear after blastokinesis and only slight growth after this results in the fully formed embryo (Fig. 4F) . The most noticeable changes being sclerotization of the mandibles, the appearance of bristles, and green colouration. The first abdominal segment is incorporated into the metathorax* by this stage so that only the notum is distinguishable. This process starts at the second blastokinetic movement and is gradual; the articulations of the metathoracic legs moving posteriorly into the first abdominal segment.

Embryonic development is now complete and the embryo resembles a first instar nymph coiled up inside the shell waiting to hatch.

*The first abdominal segment (termed the median segment) is completely fused with the metathorax in the adult. The complete fusion of the median segment is completed during nymphal life so that the first visible abdominal segment in the adult is therefore actually the second.

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S U M M A R Y

In Clitarchus hookeri the embryo develops fully on the edge of the yolk and does not become invaginated into it. Blastokinesis consists essentially of two movements, a lateral rotation through 180° followed by overgrowth of the yolk by the body walls. Segmental appendages develop into the mouthparts, legs, cerci, and (in the female) the valvifers. The other abdominal appendages are resorbed soon after they appear, except the pleuropodia on the second segment which are retained for most of the embryonic life. No appendages could be observed on the segment between the mandibles and maxillae.

DISCUSSION

Only one difference was noted between the initial formation of the embryo in C. hookeri and other Phasmidae. The blastoderm is completed before the embryo forms in other Phasmidae, whereas in C. hookeri embryo development proceeds before completion of the blastoderm is observed.

Blastokinesis resembles that in the other Phasmids Carausius morosus (Thomas 1936), Clitumnus extradentatus (Bergerard 1958), and Bacillus libanicus (Moscona 1950). The same type of blastokinetic movements occur in general in the Orthoptera (Johannsen and Butt 1941).

Roonwal (1936) found that blastokinesis in Locusta is due to movement of the living embryo, but in C. hookeri the endochorion prevents observation. However, the last blastokinetic movement is undoubtedly due to growth in Phasmids: it is a simple overgrowing of the yolk by the body wall. Phasmids then exhibit the second type of blastokinesis of Imms (1960), where the embryo is not invaginated and the embryonic envelopes form as overfolds.

Although dorsal closure of the embryo over the yolk is common to other insects, the rupture of the embryonic envelopes and overgrowth of the yolk differ. In Phasmids the embryo ruptures through both the amnion and serosa which are then left enclosing the yolk; this is common to the Orthopteroidea (Johannsen and Butt 1941) and has also been described by Mellanby (1935) for Rhodnius prolixus.

Development of the mouthparts and legs from segmental appendages is well established in the Arthropoda. Formation of the female external genitalia from the 8th and 9th appendages is also usual in other insects (Snodgrass 1933, Imms 1960). In Phasmids the embryonic retention of the second abdominal appendages (pleuropodia) appears to be usual. Normally, however, in the many other insects that do retain abdominal appendages, these are the first pair (Imms 1960), but the retention of the second in Phasmids may perhaps be correlated with the incorporation of the first abdominal segment into the metathorax.

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The function of the pleuropodia is unknown in Phasmids. Slifer (1937) found that they produced an enzyme involved with the dissolving of the calcareous serosal cuticle in Melanopsis differentialis. Among Phasmids calcium transfer has been demonstrated from the exochorion to the egg during embryonic development in Donusa prolixa (Pantel 1919) and Bacillus libanicus (Moscona 1948, 1950b), however, no calcium transfer occurs in the egg of C. hookeri (Stringer, 1968) so that here the pleuropodia cannot be concerned with calcium transfer.

A C K N O W L E D G E M E N T S

This work was undertaken as part of a M.Sc. thesis, and the Author would like to record his thanks to Associate-Professor J .G . Pendergrast and Mr D.R. Cowley for their supervision, Mr J. Kerr for invaluable cytological advice and Dr Ann Chapman for critically reading the manuscript.

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