Mesoderm formation in a springtail, Tomocerus ishibashii Yosii (Collembola : Tomoceridae)

Int. J. Insect Morphol. & Embryol., Vol. 20, No. 6, pp. 283-290, 1991 Printed in Great Britain

0020-7322191 $3.00 + .00 PergamonPressplc

M E S O D E R M FORMATION IN A SPRINGTAIL, T O M O C E R U S I S H I B A S H I I YOSII (COLLEMBOLA • TOMOCERIDAE)*

HIDEYUKI UEMIYAt and HIROSHI ANDO~ tCollege of Medical Care and Technology, Gunma University, Showa-Machi, Maebashi, Gunma 371, Japan ~Sugadaira Montane Research Center, University of Tsukuba, Sanada, Nagano 386-22, Japan

(Accepted 2 August 1991)

Abstract--The mesoderm formation of Tomocerus ishibashii (Collembola : Tomoceridae) is described. Mesodermal cells are formed after the beginning of the formation of the primary dorsal organ, and originate from the entire region of the embryonic area. After completion of the blastodermic cuticles, cells of mesoderm and ectoderm concentrate towards a ventral midline and form a well-defined 2-layered germ band. The manner of mesoderm formation in the Collembola is similar to that in Diplura and Myriapods, except for the Chilopoda; the mesoderm of the Thysanura s. lat. and Pterygota originates from a localized zone of the embryo. Within the Hexapoda, mesoderm formation is categorized into 2 types: Type 1--unlocalized origin, in the Collembola and Diplura, and Type 2-localized origin, in the Thysanura s. lat. and Pterygota. Types 1 and 2 are thought to be plesiomorphic and apomorphic, respectively. Index descriptors (in addition to those in title): Comparative embryology, Apterygota,

Entognatha, Ectognatha, Antennata, primary dorsal organ, ectoderm, germ band.

INTRODUCTION THE COLLEMBOLA is widely accepted as one of the groups that first diverged from the ancestral hexapod stock. The collembola may be important not only for discussing the evolution of the entognathous insects, but also for reconstructing the higher taxa of the Antennata. Formation of germ layers is one of the most significant problems in comparative embryology. Johannsen and Butt (1941) recognized 3 modes of m e s o d e r m (inner layer) formation within the H e x a p o d a : (1) involution, (2) outgrowth, and (3) proliferation (delamination). Although it is possible to assign the manner of m e s o d e r m formation in pterygote insects to one of the 3 modes mentioned above, those in apterygotes are fairly different between the orders (Johannsen and Butt, 1941; Jura, 1972; Anderson, 1973; Schwalm, 1988) and the interpretation of the m e s o d e r m formation in this group is highly controversial. As for the formation of m e s o d e r m in the collembola, we have Claypole's (1898) and Philiptschenko's (1912) classical studies and Jura's (1965) short description. The

*Contribution from Sugadaira Montane Research Center, University of Tsukuba, No. 133. 283

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descriptions by Claypole and Philiptschenko are apparently contradictory to Jura's, in terms of the time and manner of formation. Furthermore, these types cannot be assigned to any of the modes, which Johannsen and Butt defined. To resolve the confusion in the interpretation of the collembolan mesoderm formation, we describe details of the embryonic development of Tomocerus ishibashii from the blastoderm stage to germ band formation, re-examine the previous observations on the other collembolans, and generalize the manner of mesoderm formation in collembolans. We compare and discuss our results with the previous works on the Myriapoda and Hexapoda in relation to phylogenetic considerations. MATERIALS AND METHODS Adult T. ishibashii were collected at Sugadaira and Ueda, Nagano Prefecture. About 20 insects were kept at room temperature on pressed soil in each of several plastic cases. Females laid their eggs in May and June. Before fixation, eggs were transferred to HCl-sodium cacodylate buffer (pH 7.2), phosphate buffer (pH 7.2) and punctured with a fine needle. They were then transferred into Karnovsky'sfixative(2% paraformaldehyde + 2.5% glutaraldehyde), buffered at pH 7.2 with HCl-sodium cacodylateor 4% paraformaldehydebuffered at pH 7.2 with phosphate, and kept in this solution for 24 hr. Fixed eggs were stored in buffer solution. Fixed eggs were dehydrated by alcohol series and embedded in methacrylate resins Technovit 7100 (Kulzer) or Acrytron E (Mitsubishi Rayon). They were serially sectioned at 1-5 I~m. Sections were stained with Delafield's hematoxylinor Mayer's acid hemalum and eosin. RESULTS In the eggs of T. ishibashii, the cleavage is total and equal in earlier stages. Later, it continues in the superficial mode, and then blastoderm is formed (Uemiya, in preparation; Jura, 1972). At this stage, the egg is composed of the following kinds of cells: (1) blastoderm cells (Fig. 1), (2) yolk cells, which are widely dispersed in the yolk, and (3) primordial germ cells, which form a cluster and are situated at the center of the yolk. Just after the formation of blastoderm, a part of blastoderm becomes taller and larger (Figs 2, 3). In a short time, the nuclei of this part become situated at the bottom of each cell and the cells themselves begin to sink into the yolk to form the primary dorsal organ. Further mitoses are rarely observed in the primary dorsal organ. The organ then sinks and grows towards the internal side of the egg, to assume a spherical shape. Most of the primary dorsal organ finally sinks into the yolk and the superficial part of the organ decreases in area (Fig. 4). The differentiation of the embryonic and serosal areas soon follows the formation of the primary dorsal organ (Fig. 2). The embryonic area occupies more than half the egg surface. The serosal area is located between the embryonic area and the primary dorsal organ and superficially encircles the latter. The cells of the embryonic area proliferate, and this area itself becomes thick (Fig. 5). During the thickening, the cell arrangement in the embryonic area becomes irregular, and the area is roughly divided into an outer and an inner layer (Figs 2, 6). No differences are detected between the nuclei of these layers in the shape and stainability with hematoxylin: the outer and inner layers are the ectoderm and mesoderm (so-called "inner layer"), respectively. Now at this stage, the blastoderm differentiates into 3 regions (Fig. 2): (1) the primary dorsal organ, (2) serosa, and (3) embryo. The ectodermal cells are similar to the serosal ones both in shape and stainability with hematoxylin, but it is possible to distinguish the embryonic from the serosal areas by the presence or absence of the mesodermal layer.

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FIG. 1. Section of egg of Toraocerus ishibashii just after the beginning of formation of the primary dorsal organ. Bd = blastoderm; Ch = chorion; Y = yolk. Scale = 25 txm. FIG. 2. Section of egg of Tomocerus ishibashii showing embryonic area and extraembryonic area (primary dorsal organ and serosa). EA = embryonic area; PDO = primary dorsal organ; Ser = serosa. Scale = 100 I~m. FIG. 3. Section of early primary dorsal organ of Tomocerus ishibashii. Same stage as that of Fig. 2. PDO = primary dorsal organ. Scale = 25 p,m. FIG. 4. Section of mature primary dorsal organ of Tomocerus ishibashii. PDO = primary dorsal organ. Scale = 25 p,m.

In parallel with b l a s t o d e r m differentiation, blastodermic cuticles begin to f o r m ( U e m i y a and A n d o , 1987a). Cells of the p r i m a r y dorsal organ, serosa and e c t o d e r m (outer layer) participate in the f o r m a t i o n of cuticles, but those of the m e s o d e r m (inner layer) have no relation to cuticle formation. In step with the f o r m a t i o n of blastodermic cuticles, the e m b r y o n i c area widens and b e c o m e s thin (Fig. 7). While the nuclei of e c t o d e r m a l cells r e m a i n r o u n d in shape, the m e s o d e r m a l cells and their nuclei b e c o m e flattened. T h e s e cells n o w f o r m a well-defined layer b e n e a t h the e c t o d e r m (Fig. 7). A f t e r the c o m p l e t i o n o f the blastodermic cuticles, the chorion splits into 2 parts, and the first blastodermic cuticle is e x p o s e d to the egg surface. In parallel with the r u p t u r e of chorion, the t r a n s f o r m a t i o n of e m b r y o n i c area into the " g e r m b a n d " begins. T h e cells of the e m b r y o n i c area start concentrating t o w a r d the ventral side of the egg. T h e c o n c e n t r a t i o n of cells occurs t o w a r d the future midline of the germ band, not t o w a r d a single point. A s a result, the e m b r y o n i c area b e c o m e s thick again and the g e r m b a n d is

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~e

.....

FIG. 5. Section showing primary dorsal organ and its vicinity of Tomocerus ishibashii. Same stage as that of Fig. 2. EA = embryonic area; Ser = serosa; PDO = primary dorsal organ; Y = yolk. Scale = 25 ~,m. FIG. 6. Section of embryonic area of Tomocerus ishibashii. Mesoderm just differentiated. Note irregular 2-cell layered condition. Ec = ectoderm; Me = mesoderm. Same stage as Fig. 2. Scale = 25 ~m. Fro. 7. Section of embryo of Tomocerus ishibashii in stage of formation of blastodermic cuticles, showing flattened mesoderm and ectoderm. Same stage as that of Fig. 4. BIC1 = first blastodermic cuticle; Ch = chorion; Ec = ectoderm; Me = mesoderm. Scale = 25 Izm. Fro. 8. Section of germ band and serosa of Tornocerus ishibashii in stage after rupture of chorion. B1C1 = first blastodermic cuticle; Ec = ectoderm; Me = mesoderm; Ser = serosa. Scale = 25 Ixm.

f o r m e d , which a s s u m e s a belt-like shape e x t e n d e d a n t e r o p o s t e r i o r l y . T h e p r i m a r y dorsal o r g a n is s i t u a t e d b e t w e e n the 2 e n d s of the germ b a n d . A l s o , the m e s o d e r m a l layer b e c o m e s thicker a n d m o r e c o m p a c t a n d its nuclei r e s u m e spherical shapes (Fig. 8). T h e m e s o d e r m a l layer is clearly d i s t i n g u i s h e d from the e c t o d e r m a l layer. T h e cells of the serosa b e c o m e sheet-like in association with the c o n c e n t r a t i o n of the e m b r y o n i c area, a n d their nuclei b e c o m e f l a t t e n e d (Fig. 8). T h e serosa occupies b r o a d regions lateral to the g e r m b a n d , a n d n a r r o w regions a n t e r i o r a n d posterior to the p r i m a r y dorsal organ. This stage c o r r e s p o n d s to "Stage 1", as described by U e m i y a a n d A n d o (1987b) in the same species. Shortly, the n e u r a l g r o o v e b e c o m e s distinct a n d the e c t o d e r m a n d m e s o d e r m divide into lateral halves.

DISCUSSION F o r m a t i o n o f m e s o d e r m in the CoIlembola I n the c o l l e m b o l a , 2 m o d e s of m e s o d e r m f o r m a t i o n are r e p o r t e d . I n A n u r i d a maritima

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(Claypole, 1898) and Isotoma cinema (Philiptschenko, 1912) the mesoderm is initially formed beneath the entire blastoderm (excepting the primary dorsal organ) just after the blastoderm formation by migration and proliferation of outer cells (Philiptschenko's Table 11, Fig. 16). The mesodermal cells then concentrate to the ventral side of the egg, corresponding to the presumptive embryonic area, so that there the bilayered germ band is formed, and the unilayered serosa appears at the lateral sides of the germ band. The formation of the mesoderm in these species corresponds in time with that in T. ishibashii. The process of germ band formation is also similar in A. maritima and I. cinema, but differs from that in T. ishibashii in the following points: (1) the entire blastoderm, except for the primary dorsal organ, produces the mesoderm and becomes bilayered, and (2) the thinning of the inner layer, as observed during the formation of blastodermic cuticles in T. ishibashii, is not observed. In Tetrodontophora bielanensis (Jura, 1965), after formation of the germ band, the mesoderm arises by the inward migration and proliferation of cells of the entire germ band. As in T. ishibashii, cells of the extraembryonic area do not participate in the formation of the mesoderm. However, the mesoderm formation in T. bielanensis is delayed in comparison with that in T. ishibashii: in the former it occurs at the time of the germ band formation, in the latter at the time just after the beginning of the primary dorsal organ formation. The time designated for mesoderm formation by Jura (1965) corresponds to the time when the well-defined bilayered cell arrangement (the ectoderm and mesoderm) appears in T. ishibashii. Although there are some disagreements regarding the formation of the mesoderm, in all previously studied species and in T. ishibashii, the mesoderm originates from the entire embryonic area or germ band. Formation of mesoderm in the other Hexapoda In the dipluran, Campodea staphylinus (Uzel, 1898), about half the area of blastoderm differentiates into the embryo. At first this area is composed of irregular cells, reconstructed into 2 layers (the ectoderm and mesoderm), so that a belt-like germ band appears. Thus, mesoderm formation in C. staphylinus fundamentally resembles that in T. ishibashii. In the microcoryphian, Petrobius brevistylis (Larink, 1969), a small circular embryo or germ disc is formed at the posterior pole of the egg. Ectodermal cells of the posterior end of the germ disc proliferate, and migrate anteriorly on the dorsal surface. In Thysanura s. str. (Zygentoma), a small disc-like embryo also appears at the posterior pole of the egg (Heymons, 1897; Uzel, 1898; Wellhouse, 1954; Woodland, 1957; Larink, 1970). In Thermobia domestica and Ctenolepisma lineata (Woodland, 1957) mesoderm formation results from the proliferation and migration of cells originating from the middle region of the germ disc. Thus, mesoderm formation in the Thysanura s. str. is achieved through the proliferation and migration of cells at a restricted zone of the embryo. In the Pterygota, the mesoderm is formed by involution, outgrowth or proliferation along the midventral line of the embryo, in most cases involving the formation of a primitive groove, and occurs after the formation of the germ disc or germ band (Johannsen and Butt, 1941; Anderson, 1972a, b; Schwalm, 1988). Mesoderm formation in the Pterygota corresponds with that in the Thysanura s. lat. (Microcoryphia and Zygentoma), in that the mesoderm is derived from a restricted zone of the embryonic rudiment.

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We conclude that mesoderm formation in the Hexapoda should be first categorized into 2 types, namely Type 1: unlocalized origin (from the entire embryonic area) in the Collembola and Diplura, and Type 2: localized origin (from a restricted zone of embryonic rudiment) in the Thysanura s. lat. and Pterygota. Further, 2 types should be recognized within the latter (Type 2), i.e. Type 2.1: the mesoderm originates from the posterior region of the germ disc in the Microcoryphia, and Type 2.2: from the midventral region in Thysanura s. str. and Pterygota. The former (Type 2.1) is thought to be a primitve type of the latter, since the Microcoryphia may be regarded as a sister group of the Thysanura s. str. and Pterygota, as concluded by Machida et al. (1990), by comparing the type of cleavage. All 3 theoretical types defined by Johannsen and Butt (1941) for mesoderm formation in the Hexapoda require the midventral origin of mesoderm and can thus be applied only to the Thysanura s. str. and Pterygota. Formation o f mesoderm in the other Antennata and phylogenetic considerations In the symphylan, Hanseniella agilis (Tiegs, 1940) and the pauropodan, Pauropus silvaticus (Tiegs, 1947), a broad embryonic area is formed. The mesoderm originates

from the entire embryonic area by the inward migration and proliferation of ectodermal cells, as observed in Tomocerus ishibashii. In the diplopodan, Polydesmus abchasius (Lignau, 1911), the mesoderm is formed as in the Symphyla and Pauropoda. But in Platyrrhacus amauros (Pflugfelder, 1932), the mesoderm is formed by ingrowth from the middle region of the ectoderm. Although some interpretative confusions are recognized in the manner of mesoderm formation for diplopods, Tiegs (1940, 1947) and Anderson (1973) emphasized the resemblance between the Diplopoda and the Symphyla and Pauropoda. In the chilopodans, Scolopendra cingulata and S. dalmatica (Heymons, 1901), a small region of blastoderm differentiates into the germ rudiment. Mesoderm is formed on the dorsal surface of the entire germ rudiment. The ectoderm with the mesoderm then extends anteriorly to form the long germ band lined throughout with mesoderm. This unique manner of mesoderm formation may reflect the phylogenetic status of the Chilopoda as an isolated group among the antennate line, as Dohle (1988) postulated, and may be recognized as its autoapomorphy. As discussed previously, among the Antennata, except for the Chilopoda, we recognize 2 major types of mesoderm formation: (1) unlocalized origin (Type 1) in the Collembola, Diplura and Myriapoda, except for the Chilopoda, and (2) localized origin (Type 2) in the Thysanura s. lat. and Pterygota. Type 1 may have been directly derived from the ancestral antennate ground plan represented in the Symphyla, Pauropoda and Diplopoda, and it may be referred to as plesiomorphic. Type 2 has possibly been acquired in the Thysanura s. lat. and Pterygota close to the antennate radiations and it may be apomorphic. Thus, it may be justifiable that the Ectognatha (the Thysanura s. lat. and Pterygota) should be regarded as a monophyletic group on the basis of mesoderm formation (with Type 2 as a synapomorphy). The traditional taxon Apterygota is polyphyletic and not a natural taxon, since this older grouping requires the dissociation of the monophyletic Ectognatha (Microcoryphia, Thysanura s. str. and Pterygota). Similarly, from this point of view, the Collembola and Diplura may be regarded as a paraphyletic group against the Ectognatha. Our view is at least coincident with those of Hennig (1953, 1969), Kristensen (1975, 1989), Boudreaux (1978) and Kukalovfi-Peck (1987), mainly drawn from comparative morphology. Kristensen (1989) and Kukalovfi-Peck (1987) postulated that the Hexapoda should be divided into 3 major

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groups, (1) Ellipula (Collembola and Protura), (2) Diplura, and (3) Insecta (Ectognatha), and did not provide the entognathous insects with any status as a natural taxon. No discussions on the Protura can be presented, since we have no embryological data on this group. Acknowledgements--We thank Prof. Cz. Jura and Prof. A. Krzysztofowicz of Jagiellonian University, Krak6w, for sending us valuable articles. We also thank Dr R. Machida of the University of Tsukuba for critically reviewing the manuscript. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (02740377) to H.U.

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