Kontyc, 47(3): 367-375. September 25, 1979

Embryology of the , Protohermes grandis THUNBERG ( : ) I. Changes in External Form of the Embryo during Development

Kozo MIYAKAWA

Imafuku 1024, Kawagoe, Saitama 356, Japan

Synopsis External characteristics of the embryo of Protohermes grandis THUN- BERG during development are described and illustrated. The germ band develops on the yolk surface as typical in holometabolous eggs, but that the amnion is formed principally by growth of the posterior amniotic fold seems to be of rather hemimetabolous character. Two pairs of appendages are present on the &st abdominal segment; the median pair is the pleuropodia and the lateral is the primor- diaof the abdominal filaments (tracheal gills).

Embryonic development of the order Megaloptera has been studied on only one species, Sialis Iutaria L. belonging to the family Sialidae, by STRINDBERG(1916) and Du BOIS(1938). STRINDBERG'Swork was limited to the histological study on the germ layer formation and the organogenesis. Du BOISused Sialis eggs for the experimental study approaching the problem of determination, thus her scope was out of the general embryology except for a check of normal development. As com- pared to other insect orders, therefore, the understanding on the embryonic develop- ment of the Megaloptera is poor. Moreover, none of embryological information is available concerning the Corydalidae which is a more primitive family of the order. This paper describes the developmental stages of P. grandis as studied by ex- ternal observation and discusses its characteristics in comparison to the other insect eggs. The histological studies along the course of development will be published in future papers. Before going further, the author wishes to express his cordial thanks to Professor Dr. Hiroshi ANDO,Sugadaira Montane Research Center of the University of Tsukuba, for his kindness in critical reading through the manuscript.

Material and Method

An egg-batch composed of several hundred eggs laid by a female dobsonfly, Protohermes grandis THUNBERG,captured at night late in summer of 1976 at Okutama, Tokyo Pref., was used. The egg-batch, when oviposition, was adhered to the inner surface of a glass tube, where the mother insect had been reared. The eggs were laid at an interval of about 6 seconds. The egg-batch was kept under a condition of 100% humidity at room temperature, 21.5 to 29.5OC averaging 24°C. The egg was examined every 12 hrs, in both living and fixed conditions. For observation of living 368 Kozo MIYAKAWA Embryology of Protohermes grandis

Figs. 1-9. External form of the embryo of Protohern~esgrandis through development. 1. Embryo late in 2nd day, lateral and ventral view. ---- 2. Embryo early in 3rd day, ven- tral and lateral view. -3. Embryo at the end of 3rd day, ventral and lateral view. - 4. Embryo late in 4th day, ventral and lateral view. -5. Embryo in 6th day, ventral and lateral view. -6. Embryo in 7th day, ventral and lateral view. -7. Embryo in 8th day, lateral and ventral view. -8. Embryo in 9th day, lateral view. -9. Embryo in 11th day, ventral view. A 1-10, First to 10th abdominal segment; AF, abdominal filament or tracheal gill; AL, anal lobe; AM, amnion; AAMF, anterior amniotic fold; APP, anterior pole protuberence; AT, antenna1 rudiment; EY, eye; IC, intercalary or premandibular segment; LB, labial segment or rudiment; LR, labral rudiment; MD, mandibular segment or rudiment; MX, maxillary segment or rudiment; PAMF, posterior amniotic fold; PCE, protocephalon; PCO, protocorm; PP, pleuro- podium; RA, abdominal rudiment; SR, serosa; T 1-3, pro-, meso-, and metathoracic segment; TLl, prothoracic leg; VCH, V-shaped line of chorion; Y, yolk. eggs, liquid paraffin was used as a medium which makes the chorion transparent, but these eggs stopped their development within a half day after preparation. Fixative used was Formalin-acetic acid-ethanol mixture and eggs were fixed during 2 days and then stored in 70% ethanol. For total observation of fixed embryos the chorion was removed. Prior to dehydration the chorion was perforated with a fine needle, and sections were made employing the ordinary paraffin technique at 6 to 8 pm thick, staining with MAYER'Shaemalaum and eosin.

Observations

1. SuperJicial character of the egg The eggs were deposited as a batch of 2 to 5 layers of eggs adhered to the substratum. At oviposition each egg is sticked at its posterior pole with a dark brownish cement substance. The egg is cylindrical with round poles and with somewhat convex ventral side. It is 1.25 mm by 0.53 mm in size, and has at its anterior pole a conspicuous micropylar tubercle which is a spherical protrusion with a necked base. The chorion, 370 Kozo MIYAKAWA just after oviposition, is thin, smooth, elastic and transparent, so that creamy yolk grobules are clearly observed from outside. As development proceeds, the chorion at ventral side of the egg becomes darkly brownish and opaque except a V-shaped line along the circummicropylar region which is never pigmented (Fig. 9, VCH). This change becomes apparent on the 4th day after oviposition when the germ band attains its maximal length. This V-shaped line is to be ruptured at hatching of the larva. The egg of Protohermes grandis resembles to the eggs of Sialis lutaria (Du BOB, 1938) and S. mitsuhashii (MATSUZAKIand ANDO,1977). They are cylindrical with round poles and the ventral surface is slightly convex than the dordal one. There is a conspicuous protuberance at the anterior pole of the egg. The egg of P. grandis, however, is twice as large as that of S. lutaria. The chorionic characteristics are different between both species as follows: In P. grandis the chorion is thin and smooth whereas in S. lutaria and S. mitsuhashii it is thick and coarse with densely distributed minute protrusions on its surface. Du BOISmentioned that in S. lutaria, when hatching the chorion ruptures along a line which approximately corresponds to the contour of the "calotte micropylaire". In P. grandis this line becomes visible as the V-shaped non-pigmented line at the longest germ band stage.

2. Changes in external form of the embryo Stage 1. (1st day after oviposition): In this stage, the maturation division, the fertilization and the cleavage undergo. The histological description of this stage will be given in a future paper. Stage 2. (2nd day after oviposition, Fig. 1): Late in 2nd day after oviposition the germ band composed of a pair of protocephalic lobes and a protocorm has appeared along the ventral midline on the yolk mass. The protocephalic lobes lie immediately posterior to a slight concave region of the yolk mass at the anterior pole of the egg. The caudal end of the protocorm which is rectangular in shape occupies the postero-ventral corner of the egg. The very early stage of the germ band formation was overlooked in the present observation. The protocorm is surrounded by a groove which is deepest at its posterior end. The outer margin of this groove is the amniotic fold. Stage 3. (3rd day after oviposition, Figs. 2, 3): The germ band increases in length and decreases in width. The metamerism commences in the gnathal and thoracic regions, whereas in the abdominal region it begins later when the germ band attains its maximal length. The neural groove becomes apparent along the midline of the germ band. The germ band at longest stage occupies the ventral side, posteri- or pole and posterior 116 of the dorsal side of the egg. At this stage three gnathal, the mandibular, maxillary and labial anlagen and three thoracic leg anlagen are formed. In the meanwhile the antenna1 and labral anlagen develop. The labral rudiment is a weak flat lobe, not showing a paired structure from a superficial view. The intercalary or premandibular segment becomes apparent between the proto- Embryology of Protohermes grandis 371 cephalic area and the mandibular segment, but without any appendage rudiments, and the abdominal region shows demarcations of first 5 or 6 segments, but those of caudal segments are not yet clear. At the beginning of this stage the posterior amniotic fold of the germ band is vigorously growing anteriorwards through a posterior end of the thoracic region. The anterior and lateral amniotic folds, on the contrary, are less developed. Con- sequently a large part of protocephalic and gnatho-thoracic region is naked. These amniotic folds grow and meet together at a gnathal region by the end of this stage. Thus, the germ band is completely covered by the amnion and the serosa. Stage 4. (4th day after oviposition, Fig. 4) : The germ band or embryo increases in width except in the cephalic region, and the width of thoracic region expands to more than three fourths of the egg diameter. Each of maxillary and labial rudi- ments, near their distal ends, bears a palpus pointing lateralwards. The leg rudi- ments extend medio-posteriorwards and reach the posterior border of the following segment. The abdomen becomes 10-segmented and the neural groove becomes apparent along the ventral midline of the embryo. The pleuropodia appear in the first abdominal segment. In dissected specimens and in section, the stomodaeum grows antero-dorsalwards and the proctodaeum begins to develop. During this stage the chorion becomes pigmented as mentioned above. Stage 5. (5th day after oviposition): The embryo widens and revolution of the embryo begins in the posterior end of the abdomen. At this stage, the embryo as a whole does not move in the egg, locating on the ventral surface of the yolk mass. The antenna1 anlagen become 3-segmented. Each maxillary anlage bears an additional branch (rudiment of the lacinia) on a median side of the rudiments of galea. The abdominal segments have two pairs of swellings. The median ones im- mediately lateral to the ventral nerve cord are small and homotopous with the pleuropodia. These median swellings later disappear. The lateral swellings are conspicuous structures located between the median swellings and tracheal pits, and they later give rise to the abdominal filaments or tracheal gills. In the posterior abdominal segments, the formation of these swellings is retarded. The tracheal pits appear in the mesothoracic and first 8 abdominal segments and localize in antero-lateral to the anlagen of abdominal filaments. Stage 6. (6th day after oviposition, Fig. 5): The embryo more widens especially in the abdominal region and occupies nearly a ventral half of the egg. As revolution of the embryo proceeds the 9th abdominal segment is located at the posterior pole of the egg and the 10th abdominal segment bends ventrally, therefore it comes ventral to the 8th abdominal segment. The morphogenetic movement of the cephalo-gnathal region begins as shortening especially in the gnathal region. The labial rudiments move anteriorwards between the maxillary ones. Therefore the labial rudiments become located between maxil- lary ones, and the prothoracic segment now immediately follows the maxillary 372 Kozo MIYAKAWA segment. The distal ends of rudimental metathoracic legs extend beyond the third ab- dominal segment, and the tips of all rudiments are bifurcated. The anlagen of the abdominal filaments on the first 8 abdominal segments rapidly develop, whereas the median swellings regarded as serially homologous with the pleuropodia not more develop. The pleuropodia on the first abdominal segment are, in contrast, observ- able as a pair of conspicuous protrusions with a necked base. The telson bears a pair of big anal lobes and their distal end reaches a level of the 5th abdominal segment. Stage 7. (7th day after oviposition, Fig. 6): The embryo becomes widest and then rapidly accomplishes revolution. This stage is characterized in that the embryo increases notably in length of abdomen and meanwhile the curvature of the abdomen turns ventrally (Compare Figs. 6 and 7). So that the 4th and 5th abdominal segments become located at the posterior pole in the egg, and the 10th abdominal segment goes anteriorwards between thoracic legs, and the caudal end of the abdomen attains the level of mesothorax where it meets with the tips of the maxillae. The abdomen, through this change, becomes cylindric. This dorsal closure begins first at the posterior region of the abdomen and then proceeds anterior- wards. The yolk mass is nearly enclosed by the dorsal wall of the embryo, but the dorsal closure is not yet completed at this time. Basic form of the larval head is established. The eye rudiments appear, and the antennae and all the mouth parts elongate and point medio-posteriorwards. The metathoracic legs attain the posterior pole of the egg. The pleuropodium becomes a thick and short structure with a small protrusion on its distal end. A pair of the lobe-like abdominal filaments are observed on each of the first 8 abdominal segments. In the first abdominal segment, they localize laterally to the pleuropodia. The embryonic membranes rupture and are taken into the yolk from the dorsal part of thoracic region of the embryo. Stage 8. (8th day after oviposition, Fig. 7): No further change is observed in the proportion and the position of the embryo in the egg. Remarkable changes are the development of eyes, abdominal filaments and anal appendages or pygopods. The eyes appear as notable swellings on latero-posterior corners of the head. It is composed of 7 ommatidia or facettes arranged in a depressed hexagon, six of which at each angle of, and one in the center of the hexagon, as observed in the embryos of primitive dragonflies (ANDO,1957). At first they are colourless and as development advances they change into red-brownish except the most posterior one. Then the eye develops as consisting of 6 ommatidia. The thoracic leg rudi- ments are segmented into the coxa, femur, tibia and tarsus with two tarsal claws. The pleuropodia decrease in height. The abdominal filaments elongate nearly twice as long as the abdominal segment. The living embryos show temporary move- ments of their body parts. Stage 9. (9th to 13th day after oviposition, Figs. 8, 9): This is the final stage Embryology of Protohermes grandis 373 of the embryonic development. At the end of 10th day after oviposition, the definitive integument is completed by embryonic moulting. The eyes become heavily pigmented. The antennae are 3-segmented and bear a seta on a distal part of the second segment. The mandibles become sclerotized and pigmented at their distal end which is bifurcated with acute tips. The maxillary palpi are definitively segmented with three joints. The thoracic legs bear a set of setae at a distal part of the femur. The abdominal filaments elongate rapidly and extend nearly three times as long as an abdominal segment. In living embryos it is readily observed that the old cuticular skin is thrown, and a space appears between the old skin and the newly developing integument (Fig. 9). The embryos hatch out in the latter half of 13th day from the oviposition. When hatching the whitish V-shaped line at the anterior pole of the egg shell, as previously mentioned, is broken and the new larva hatches out anteriorwards with the peristaltic movement of the thoracic and abdominal regions. After hatching the larvae actively walk around on the substratum with a posture, the tip of their abdomen pointing dorsalwards, for a considerable time. When dropped into water, they at first do not walk, but swim with repeated bendings of their abdomen.

Discussion Duration of the embryonic development of Protohermes grandis differs from that of SiaZis lutaria observed by Du Bors (1938), but processes of both species, rela- tive to the total duration of embryonic period agree well with each other as shown in Table 1. In both species the amnion formation is retarded till the germ band has exhibited the segmentation except the developing abdominal region. Similar late formation of the amnion was observed in Chrysopa perla (Neuroptera, BOCK,

Table 1. Developmental sequence indicated by day and index per total embryonic period. P. grandis S. Iutaria Events in embryonic development (Du BOIS, 1938) Days Index Days Index Oviposition 0 (0) 0 (0) Protocephalon and protocorm differentiate 2 (15) 1 (11) Appendage rudiments appear/Amnion completed 3 (23) 2 (22) Embryo longest/Segmentation of abdomen 4 (31) 3 (33) Abdominal filaments begin to grow 6 (46) 4 (44) Eyes begin to form 7 (54) 5 (55) Dorsal closure occurs 8.5 (65) 6 (67) Legs attain their definitive length 10 (77) 7 (78) Embryonic moulting occurs 11 (85) ? Hatching 13 (100) 9 (100) Egg size 1.25~0.53rnm 0.65x0.25mm Average temperature 24°C 19°C 3 74 Kozo MIYAKAWA

1939) in which the amnion is completed after the appearance of appendage rudi- ments. In these megalopteran and neuropteran embryos the amnion is formed principally by growth of the posterior amniotic fold. This mode of amnion for- mation closely resembles to that observed in Panorpa pryeri (ANDO, 1970) and Pa. japonica (ANDOand MIYAKAWA,unpublished) though in Panorpa the completion of the amnion preceeds the segmentation of the germ band. That the posterior amniotic fold is dominant as mentioned above may be of hemimetabolous character rather than holometabolous one. In P. grandis embryo, two pairs of appendage rudiments develop on the first abdominal segment. The median pair is the pleuropodia and the lateral pair is rudiments of the abdominal filaments or tracheal gills. The pleuropodia become apparent from the 4th day after oviposition (Fig. 4), and the abdominal filaments differentiate considerably later from the lateral swellings (Figs. 5-9). The formation of two pairs of appendages on the first abdominal segment is rare in insect embryos. P. grandis embryo, therefore, may offer an interesting case related to the problem on the homology of abdominal appendages with thoracic legs in . SNODGRASS(1935) mentioned that in pterygote larvae some of abdominal appendages originate from the limb-like rudiments of the embryo and these are parts at least of true segmental appendages. On the contrary, HINTON(1955) proposed his theory, principally on the base of Dollo's law, that the abdominal appendages (prolegs) of holometabolous larvae are not homologous with the thoracic legs, but are secondary adaptive structures. MATSUDA(1976), however, insists that abdomi- nal appendages are homologous with thoracic legs as implied by BERLESE,denying HINTON'Stheory on the base of heterochrony which occurs in organogenesis during embryonic and postembryonic developments. Recently ANDOand HAGA(1974) demonstrated in the embryo of Pa. pryeri (Mecoptera) a pair of styliform appendages which develop along the median ventral line on each of the first 8 abdominal segments and persist into larval stage. In P. grandis such a series of appendages is lacking. Neither pleuropodia nor abdomi- nal filaments of P. grandis are homotopous with styliform appendages of Pa. pryeri. These facts suggest that there are at least 3 possible series of abdominal appendages in insect embryos, (1) lateral type as represented by the megalopterous abdominal filamens, (2) latero-ventral type as represented by the pleurpodia of P. grandis, Stenopsyche griseipennis (Trichoptera, MIYAKAWA,1973, 1975) and other insect embryos, and (3) ventral type as represented by the styliform appendages of Pa. pryeri (ANDOand HAGA,1974). As to the homology of these abdominal appendages with the thoracic legs, however, the present study cannot give any reasonable ex- planation. Embryology of Protohermes grandis 375

References

ANDO,H., 1957. A comparative study on the development of ommatidia in Odonata. Sci. Rep. Tokyo Kyoiku Daigaku, Sect. B., 8: 174-216. ----- 1970. Insect Embryology. In Systematic zoology, 7-IIIa, pp. 37-130, Nakayama Shoten, Tokyo. (In Japanese.) ANDO,H., and K. HAGA,1974. Studies on the pleuropodia of Embioptera, Thysanoptera and Mecoptera. Bull. Sugadaira biol. Lab., Tokyo Kyoiku Univ., 6: 1-8. BOCK,E., 1939. Bildung und Differenzierung der Keimblatter bei Chrysopa perla (L.). Z. Morph. Oekol. Tiere, 35: 615-702. Du Bo~s,A. M., 1938. La determination de l'kbauche embryonnaire chez Sialis lutaria L. (Megaloptera). Rev. Suisse Zool., 45: 1-92. HINTON,H. E., 1955. On the structure, function, and distribution of the prolegs of the Panor- poidea, with a criticism of the Berlese-Imms theory. Trans. R. ent. Soc. London, 106: 455-545. MATSUDA,R., 1976. Morphology and evolution of the insect abdomen. 534 pp. Pergamon Press, Oxford. MATSUZAKI,M., and H. ANDO,1977. Ovarian structures of the adult alderfly, Sialis mitsuhashii OKAMOTO(Megaloptera: Sialidae). Intern. J. Ins. Morphol. & Embryol., 6: 17-29. MIYAKAWA,K., 1973. The embryology of the caddisfly Stenopsyche griseipennis MACLACHLAN (Trichoptera: Stenopsychidae). I. Early stages and changes in external form of embryo. Kontyii, 41 : 413-425. 1975. Ditto. V. Formation of alimentary canal and other structures, general con- sideration and conclusion. Zbid., 43: 55-74. SNODGRASS,R. E., 1935. Principles of insect morphology. 667 pp. McGraw-Hill, New York & London. STRINDBERG,H., 1916. Hauptziige der Entwicklungsgeschichte von Sialis lutaria L. Zool. Anz., 46: 167-185. Bibliography of the Neuropterida

Bibliography of the Neuropterida Reference number (r#): 8266

Reference Citation: Miyakawa, K. 1979 [1979.??.??]. Embryology of the dobsonfly, Protohermes grandis Thunberg (Megaloptera: Corydalidae), I. Changes in external form of the embryo during development. Kontyû 47:367-375.

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