Embryogenesis of the Mayfly Ephemera Japonica Mclachlan

JOURNAL OF MORPHOLOGY 234:97–107 (1997)

Embryogenesis of the Mayﬂy Ephemera japonica McLachlan (Insecta: Ephemeroptera, Ephemeridae), With Special Reference to Abdominal Formation

KOJI TOJO* AND RYUICHIRO MACHIDA Institute of Biological Sciences, University of Tsukuba, Tsukuba, Ibaraki 305 Japan

ABSTRACT Embryogenesis of the mayfly Ephemera japonica is described, with special reference to the abdominal morphogenesis. Cleavage is of the typical superficial type. The germ disc is formed by the cell concentration of the embryonic area broadly defined at the posterior half of the blastoderm. The embryo undergoes embryogenesis of the typical short germ type. Blastoki- nesis in which the extensive and deep invagination of the embryo is involved is similar to those of odonatans and plecopterans. In the longest embryo stage, the abdomen is folded and is divided into four regions (regions I–IV, from anterior to posterior). All the first to eleven segments are derived from regions I and II. Regions III and IV fuse together to form the proctodaeum. This manner of abdominal formation may be regarded as basic in pterygote insects, because a similar manner is found in another palaeopteran group, the odonatans. The caudal filament has been interpreted as the elongation of the eleventh abdominal tergum, but it is now revealed that the caudal filament originates from the posterior extremity of region IV and that its origin is away from the eleventh segment, with regions III and IV or the proctodaeum interposing between. Thus, it is concluded that the caudal filament should be correlated not to the eleventh abdominal segment but to the telson. J. Morphol. 234:97–107, 1997. ௠ 1997 Wiley-Liss, Inc.

The Ephemeroptera is an important group In the first part of our studies, we outline in attempting to discuss the phylogeny of the embryogenesis of the species, with spe- pterygote basal clades, which remains con- cial reference to abdominal morphogenesis. troversial (cf. Hennig, ’69; Kristensen, ’75; Boudreaux, ’79), for the Ephemeroptera is MATERIALS AND METHODS widely accepted as one of the pterygote rep- The mature females of Ephemera japonica resentatives closest to early pterygote ances- McLachlan (suborder Schistonota, family tors. The Ephemeroptera is also important Ephemeridae) were collected at the Kakuma for understanding the ground plan of the River and at a branch of Karasawa River, Pterygota as well as for speculation on hexa- Sanada, Nagano Prefecture, Central Japan, pod evolution. in July of 1993 to 1996, and eggs were ob- For phylogenetical discussion, the com- tained in the laboratory. The eggs were incu- parative embryological approach is one of bated in water at room temperature the most promising methods. The embryo- (20 Ϯ 2°C). They were fixed with alcoholic genesis of Ephemeroptera has been studied Bouin’s fluid (saturated alcoholic solution of by Joly (1876), Heymons (1896a,b,c), Mur- phy (’22), Ando and Kawana (’56), Wolf (’60), Bohle (’69), and Tsui and Peters (’74), but Contract grant sponsor: Showa Seitoku Foundation; Contract details of the ephemeropteran embryology grant sponsor: Ministry of Education, Science and Culture of still remain fragmented. We have started a Japan; Contract grant number: 08640881. *Correspondence to: Koji Tojo, Sugadaira Montane Research comparative embryological study of the Center, University of Tsukuba, Sanada, Nagano 386-22, Japan. Ephemeroptera using Ephemera japonica. E-mail: [email protected].

௠ 1997 WILEY-LISS, INC. 98 K. TOJO AND R. MACHIDA picric acid:formalin:acetic acid ϭ 15:5:1) at disc. The anterior cells become more flat- room temperature for 24 h. Embryos were tened and form the future serosa. dissected in Ephrussi-Beadle solution (7.5 g Stage 4: Pear-shaped embryo (Fig. 1D,DЈ) NaCl ϩ 0.35 g KCl ϩ 0.21 g CaCl2/1,000 ml distilled water) with fine forceps and then The germ band starts to elongate back- fixed. The fixed eggs and embryos were wards. It assumes a pear shape, and the stored in 70% ethyl alcohol. anterior broad protocephalon and posterior The eggs or embryos were processed into protocorm differentiate. methacrylate resin Technovit 7100 (Kulzer) Stage 5: Start of invagination of germ band or water miscible epoxy resin Quetol 651 Ј (Nisshin EM) sections of 1–2 µm thickness, (Anatrepsis I) (Fig. 1E,E ) in accordance with Machida et al. (’94a,b). The germ band further elongates, and its Sections were stained with Delafield’s hema- caudal end starts to proceed into the yolk. At toxylin and eosin or, with Schiff’s reagent the same time, the amnion starts to be pro- (nucleal or PAS reaction), Mayer’s acid hem- duced from the embryonic margin, resulting alum and fast green FCF. in the start of the amnioserosal fold forma- For scanning electron microscopy (SEM), tion. This amnioserosal fold is derived mainly the fixed embryos were sonicated for a few from the posterior area, the anterior amnio- seconds with an ultrasonic cleaner, dehy- serosal fold being not well developed. drated in a graded ethyl alcohol series, and Stage 6: S-shaped embryo (Anatrepsis II) then transferred to acetone. The embryos Ј were dried in a critical-point drier, coated (Fig. 1F,F ) with gold, and observed under a JSM-T200 With active cell proliferation, the embryo scanning electron microscope (JEOL, To- further elongates and deeply invaginates into kyo). the yolk. The caudal end of the embryo reaches the middle of the egg long axis, and RESULTS the embryo acquires an S-shape. At this The egg period of Ephemera japonica stage, the posterior amnioserosal fold ex- ranges from 15–17 days at room tempera- tends anteriorly to the cephalic level, and ture (20 Ϯ 2°C). Herein we describe the em- anatrepsis is completed (Fig. 2). The ventral bryonic development, dividing it into 13 side of the embryo is completely covered by stages, with special reference to abdominal the amnioserosal fold or the amnion. The development. The most important stages for inner layer or mesoderm differentiates all the abdominal morphogenesis are stages over the dorsal side of the embryo (cf. Fig. 3). 7–10. Stage 7: Longest embryo (Fig. 1G) The embryo further elongates, with its Stage 1: Egg cleavage (Fig. 1A) posterior half bending three times, and it Egg cleavage is of the typical superficial type. The first five cleavages are synchro- nized, each cleavage occurring at intervals of approximately 9 h. Fig. 1. Ephemera japonica. Successive stages of embryonic development. Lateral views. I–IV, abdominal Stage 2: Blastoderm formation (Fig. 1B) regions I–IV; AbS4, 10, 11, fourth, tenth, and eleventh Cleavage nuclei arrive at the egg periph- abdominal segments, respectively; Am, amnion; An, antenna; BdC, blastoderm cell; CE, compound eye; Ce, ery after eight cleavages, and the syncytial cercus; CF, caudal filament; CN, cleavage nucleus; ET, blastoderm (blastema) is formed. Soon the egg tooth; GD, germ disc; HL, head lobe; Lb, labium; Md, cell membrane appears between the blasto- mandibile; Mx, maxilla; Oc, ocellus; Pc, protocephalon; derm cells, and the blastoderm (blastoderm Po, protocorm; PsAm, presumptive amnion; SDO, secondary dorsal organ; Se, serosa; TL1, proleg; Y, yolk. Bar ϭ s. str.) is completed. 50 µm. A: Stage 1 (egg cleavage). B: Stage 2 (blastoderm formation). C: Stage 3 (germ disc formation). D,DЈ: Stage 3: Germ disc formation (Fig. 1C) Early (D) and late (DЈ) stage 4 (pear-shaped embryo). E,EЈ: Early (E) and late (EЈ) stage 5 (start of invagina- Even in the newly formed blastoderm, the tion of germ band). F,FЈ: Early (F) and late (FЈ) stage 6 posterior half embryonic and anterior half (S-shaped embryo). G: Stage 7 (longest embryo). H: Stage 8 (segmentation of embryo). I: Stage 9 (procto- extraembryonic areas are distinguishable daeum formation). J: Stage 10 (revolution). K: Stage 11 (Fig. 1B). The posterior cells concentrate at (postrevolution I). L: Stage 12 (postrevolution II). M: the posterior pole of the egg to form the germ Stage 13 (postrevolution III). EMBRYOGENESIS OF MAYFLY 99

Figure 1 100 K. TOJO AND R. MACHIDA

Fig. 2. Ephemera japonica. Am, amnion; AmSeF, pleted. Bar ϭ 50 µm. B: Enlargement. The amnioserosal amnioserosal fold; An, antenna; Ch, chorion; Em, em- folds fuse with each of those beneath the embryo at the bryo; Md, mandible; Se, serosa. A: Sagittal sections of level of labrum (arrow), and anatrepsis is completed. egg, at late stage 6, in which anatrepsis is just com- Bar ϭ 10 µm. acquires its maximum length, reaching near soon enclosed by the definitive dorsal clo- the anterior egg pole. With the bendings, the sure of region II (Figs. 4A, 9B,BЈ,C). posterior half of embryo or the future abdo- The caudal filament develops at the apex men is folded and divided into four regions of region IV (Figs. 4B, 7A,B, 8, 9). A pair of (regions I–IV from the anterior to posterior cerci develops at the eleventh abdominal [Fig. 3A]). In cross-section, regions I and II segment, which is the extremity of region II. and regions III and IV are respectively con- With the progression of definitive dorsal clo- nected to each other by the amnion (cf. Fig. sure, the cerci move from the original ventro- 3B), and all of the regions are dorsally lined lateral to the dorsolateral position and reach with the inner layer (Fig. 3B). their definitive position at the same level as the caudal filament (Figs. 7, 9B vs. Figs. 8, Stage 8: Segmentation of embryo (Fig. 1H) 9C). The segmentation of embryo commences. It proceeds from the head to the rear, and Stage 10: Revolution (Katatrepsis) (Fig. 1J) simultaneously appendage rudiments ap- The amnioserosal fold tears near the la- pear in the cephalic and thoracic segments. brum, and the embryo appears on the egg In regions I and II of the abdomen, now surface. The embryo, which is slightly short- divided into four regions, the first to fifth ened temporarily, moves along the ventral and the sixth to eleventh abdominal seg- surface of the egg towards the anterior pole, ments, respectively, develop (Figs. 5A, 9A). with its head to the traveling direction, and Late in this stage, neuroblasts and neuro- the antero-posterior axis of the embryo re- piles can be clearly observed in all the first verses. to eleventh abdominal segments (Fig. 5A,B; cf. Fig. 6A,B). In contrast, no attributes of a segmental nature can be found in regions III Stage 11: Postrevolution I (Fig. 1K) and IV. The serosal cells are condensed at the antero-dorsal part of the egg to form the Stage 9: Proctodaeum formation (Fig. 1I) secondary dorsal organ just posteriorly to The appendages of the head and thorax the head. With the progressive condensation are enlarged and segmented. Regions III and withdrawal of serosal cells, the amnion and IV of the abdomen fuse with each other replaces the serosa and finally spreads over dorsally to form the proctodaeum (Figs. 3B, the dorsal yolk as a provisional dorsal clo- 4A, 9B,BЈ). Then the proctodaeum itself is sure. EMBRYOGENESIS OF MAYFLY 101

Figs. 3, 4. Ephemera japonica. Sections of eggs at these regions on this plane have already fused with each stages 7 and 9. other. Regions I–IV are found to be dorsally lined with the inner layer. Bar ϭ 20 µm. Fig. 3. Eggs of stage 7. I–IV, abdominal regions I–IV; Am, amnion; Ch, chorion; Em, embryo; IL, inner layer; Fig. 4. Eggs of stage 9. I–IV, abdominal regions I–IV; Pd, proctodaeum; Se, serosa; Th, thorax; Y, yolk. A: Ce, cercus; CF, caudal filament; Ch, chorion; Pd, procto- Sagittal section. Embryo acquires its maximum length daeum; Se, serosa; SeC, serosal cuticle; Th, thorax. with its posterior half bending three times. With bend- Bars ϭ 25 µm. A: Cross-section at the eleventh abdomi- ing, the posterior half of the embryo or the future abdo- nal segment. Regions III and IV of the abdomen or the men is folded and divided into four regions (regions proctodaeum is enclosed by region II. B: Cross-section at I–IV). Bar ϭ 50 µm. B: Cross-section approximately the base of the caudal filament and cerci. From A and B, through a–aЈ level in A. In this stage, regions I and II it is clear that the caudal filament develops at the apex and regions III and IV are, respectively, connected with of region IV and that a pair of cerci develops at the each other by the amnion, but between region III and IV eleventh abdominal segment, which is the extremity of the amniotic communications are not found, because region II.

Stage 12: Postrevolution II (Fig. 1L) The larval cuticle is secreted, and a sclero- The secondary dorsal organ which formed tized egg tooth is visible on the frons. at the previous stage sinks into the yolk and disappears. The appendages of the head and DISCUSSION thorax further develop. The definitive dorsal General aspects of embryogenesis closure further proceeds anteriorwards. A The germ disc of Ephemera japonica is pair of cerci and the caudal filament elon- formed with a concentration of cells at the gate and become segmented. Late in this stage, compound eyes and three ocelli can be embryonic area that is broadly defined at clearly observed. the posterior half of the blastoderm. A similar manner of germ disc formation is ob- Stage 13: Postrevolution III (Fig. 1M) served in other ephemeropterans (i.e., The definitive dorsal closure is completed, Ephemera strigata [Ando and Kawana, ’56], and the embryo acquires its definitive form. Baetis rhodani and B. vernus [Bohle, ’69]), 102 K. TOJO AND R. MACHIDA

Figures 5–6 EMBRYOGENESIS OF MAYFLY 103 and this manner of germ disc formation may divided into four regions, I–IV. Regions III be regarded as an important characteristic and IV fuse together to form the procto- of ephemeropteran embryogenesis. daeum; the ventral and dorsal walls, respec- The embryo of Ephemera japonica can be tively, originate from regions III and IV. A categorized as the typical short germ type similar manner of proctodaeum formation is (cf. Krause, ’39), characterized by the sequen- found in another palaeopteran group, the tial proliferation of segments from the ante- Odonata (Ando, ’62), and this type of procto- rior to posterior. Because the short germ daeum formation, in which the proctodaeum type dominates in primitive pterygotes (e.g., is formed by the fusion of two belt-like rudi- Odonata [Ando, ’62] Plecoptera [Kishimoto ments, may be regarded as one of the most and Ando, ’85]) as well as in ectognathan basic in Pterygota. apterygotes (cf. Sander, ’84), this may be The proctodaeum formations in neopteran regarded as an ancestral feature. As a result orders may be categorized as this type, such of anatrepsis and elongation of the germ as the hemipterans Pyrrhocoris apterus band, the embryo of Ephemera japonica ac- (Seidel, ’24) and Oncopeltus fasciatus (Butt, quires an S-shape, as observed in other ’49), the homopteran Pyrilla perpusilla primitive pterygotes such as an odonatan (Sander, ’56), the mecopteran Panorpa pry- Epiophebia superstes (Ando, ’62) and a ple- eri (Suzuki and Ando, ’81), the trichopteran copteran Kamimuria tibialis (Kishimoto and Stenopsyche griseipennis (Miyakawa, ’75), Ando, ’85), and this type of blastokinesis and the lower lepidopterans Endoclita signi- may be considered to be plesiomorphic within fer (Kobayashi et al., ’81) and Neomicrop- the pterygotes. teryx nipponensis (Kobayashi and Ando, ’88). It has been confirmed that in Ephemera In these insects, region IV has been re- japonica the serosal cuticle is not secreted garded as amniotic in origin, because of its until the completion of anatrepsis—that is, lack of inner layer, and their proctodaeum until the embryo is ventrally covered by the has been recognized to be dual in origin (i.e., amnioserosal fold and the entire egg surface is occupied by serosa. This may be favorable originating from the dorsal wall of the am- to the idea of Machida et al. (’94a) concern- nion and the ventral wall of the embryo ing the functional role of the amnioserosal proper) (Seidel (’24) observed that both the fold. They deduced that the amnioserosal dorsal and ventral walls of P. apterus procto- fold should have been acquired during ectog- daeum lack the inner layer, and he con- nathan evolution in order to secrete the sero- cluded the whole proctodaeum of this insect sal cuticle beneath the embryo that had lost to be amniotic in origin) (cf. papers men- this ability. tioned above). In light of this, it may also be said that region IV, namely the whole procto- Proctodaeum formation daeum, of the Palaeoptera (Ephemeroptera In the longest embryo stage (stage 7), the (herein) Odonata [Ando, ’62]) is embryonic abdomen of Ephemera japonica is folded and proper in origin, because the inner layer is found to be segregated also on region IV, namely on the entire proctodaeum. Accordingly, we may recognize the difference in proctodaeum formation, concerning Figs. 5, 6. Ephemera japonica. Sagittal sections of the differentiation of the inner layer, be- the embryo at stage 8 and egg at stage 11. tween the palaeopterans and neopterans. Fig. 5. I–IV, abdominal regions I–IV; AbS1, 6, 9–11, However, we suggest that the difference is first, sixth, and ninth to eleventh abdominal segments; not so essential as to specifically designate CF, caudal filament; Nb11, neuroblast of eleventh ab- the partially different origin of the procto- dominal segment; Sd, stomodaeum; Th3, metathorax. A: Embryo just before katatrepsis at stage 8. Bar ϭ 50 µm. daeum. We believe that the difference can be B: Enlargement of abdomen. A neuroblast is clearly explained merely by the term heterochrony, observed in the eleventh abdominal segment. Bar ϭ 10 concerning the timing of the segregation of µm. the inner layer and the morphogenesis of the Fig. 6. AbS1 and 11, first and eleventh abdominal proctodaeum, or by the phrase ‘‘substitution segments; Np1, 6–11, neuropiles in the first and sixth to in the developmental process of Matsuda eleventh abdominal segments; Sd, stomodaeum; Th, tho- (’76),’’ namely the evolutional alteration of rax; Th3, metathorax; Y, yolk. A: Egg at stage 11. Bar ϭ 50 µm. B: Enlargement of abdomen. Neuropiles are the manner of mesodermal supply to the clearly differentiated in the first to eleventh abdominal wall of the proctodaeum. Since we cannot segments. Bar ϭ 10 µm. find any new bases for the amniotic nature 104 K. TOJO AND R. MACHIDA

Figs. 7, 8. Ephemera japonica. SEM micrographs of Fig. 8. Ventro-lateral view of the abdomen, more abdomens at stage 9. developed than that in Fig. 7. The cerci move from their original ventro-lateral to the dorso-lateral position, and Fig. 7. AbS1–11, first to eleventh abdominal seg- they reach their definitive position at the same level as ments; Ce, cercus; CF, caudal filament; Pd, procto- the caudal filament. AbS4–11, fourth to eleventh abdomi- ϭ daeum; TL3, metaleg. Bars 10 µm. A: Ventro-lateral nal segments; Ce, cercus; CF, caudal filament; Pd, proc- view of the abdomen. B: Enlargement from a different todaeum. Bar ϭ 30 µm. angle. It is clear that the origins of cerci and the caudal filament are different. EMBRYOGENESIS OF MAYFLY 105

Fig. 9. Ephemera japonica. Diagrammatic representation of abdominal development. I–IV, abdominal regions I–IV; AbS1–11, first to eleventh abdominal segments; Ce, cercus; CF, caudal filament; Pd, proctodaeum. A: Stage 8. B,BЈ: Early stage 9. In BЈ, regions III and IV are pulled apart from region II to show the proctodaeum in formation. C: Late stage 9. The proctodaeum is enclosed by the definitive dorsal closure of region II. 106 K. TOJO AND R. MACHIDA of region IV in neopterans, it is better to versity of Tsukuba. We also thank Dr. recognize the region to be of the embryo Tadaaki Tsutsumi, Fukushima University, proper in origin as well. for his invaluable suggestions and advice throughout this study. This work was sup- Caudal filament and abdominal formation ported by grants in aid from the Showa Seitoku Foundation and from the Ministry Heymons (1896a,b,c) deduced that the cau- of Education, Science and Culture of Japan dal filament is the elongation of the eleventh (08640881) to R.M. This is contribution 156 abdominal tergum. His idea has been fol- from the Sugadaira Montane Research Cen- lowed by many insect morphologists, such as ter, University of Tsukuba. Crampton (’17, ’18), Snodgrass (’35), Brinck (’57), Birket-Smith (’71), and Matsuda (’76), and has remained largely unchallenged. LITERATURE CITED In our present study, we clarify that the Ando, H. (1962) The Comparative Embryology of Odo- nata With Special Reference to a Relic Dragonfly, caudal filament of Ephemera japonica origi- Epiophlebia superstes Selys. Tokyo: Japan Society for nates from the posterior extremity of region the Promotion of Science. IV and has its origin considerably away from Ando, H., and T. Kawana (1956) Embryology of mayfly the eleventh abdominal segment, with re- (Ephemera strigata Eaton) as studied by external ob- servation. Kontyuˆ 24:224–232 [in Japanese]. gions III and IV or the precursor of procto- Birket-Smith, J. 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