Recent Advances in Insect Embryology in Japan
Edited by H. Ando and K. Miya.
ISEBU Co. Ltd., Tsukuba 1985

203

External Morphogenesis of the Embryo of
Asca laphus ramburi(N europtera, Ascalaphidae)

Akimasa KAMIY A
andHiroshi ANDO

Synopsis
External form of the developing embryos of Ascalaphus ramburi MacLachlan is observed and thenormal plate of the embryonic development is given. Micropyles are observed at each pole of the egg anda girdling or hatching line presents at the anterior pole of the egg. The large, broad ventral plate differentiates in the blastoderm and the embryo remains superficial during its entire development. The segmentation of the germ band has occurred before the inner layer formation. A large secondary dorsal organ isformed.

Introduction
The Ascalaphidae are actively flying neuropteran insects and widely distribute in theworld. In Japan four species belonging to four genera are known, and the Japanese Ascalaphidae were studied taxonomicalIy by Okamoto (1909) and autoecologically by Sonan(I938), but the embryonic development of Ascalaphidae was not studied up to this time.
Concerning the Neuroptera and Megaloptera works dealing with the embryonic development are on Chrysopa perla (Bock, 1939, 41), Sialis lutaria (Strindberg, 1915; DuBois,
1936,38), S. mitsuhashii (Suzukietal, 1981) andProtohermesgrandis (Miyakawa, 1979,
80).
In the present paper the outline of the embryonic development of Ascalaphus ramburi is
described, and the detailed studies on embryogenesis of this species will be published in thenear future.

Kamiya, A. and H. Ando

204

Material and Methods
The egg of Ascalaphus ramburi MacLachlan were collected during late May and mid Junein Kaida and Ueda in Nagano Prefecture and Shirotori in Gifu Prefecture of Japan, and they
were reared in plastic cases at room temperature.
Almost all egg-masses were oviposited on dead stems of various grasses or dead twigs oftrees in grass-fields surrounded by trees.
Observations of the developing embryos were done under living or fixed conditions. Especially at early stages of the embryonic development, living eggs were mostly used for observation. The eggs were fixed with F. A. A., Carnoy's solution or Bouin's solution. F. A.
A. and Bouin's solution were heated at 60°C to 70°C before use.
When the eggs areobserved, the outer layer of the chorion was dissolved by 4% antiformin.

Results
1.

External Structure of the Egg

The eggs of Ascalaphus ramburi are creamly-white in color and oval in shape, about 2 mm
in length and 1.3 mm in width. Eggs are deposited in rows upon grass stems or twigs, theventral side of the egg being laid over these substrata. Though in the American Ascalaphidae
modified eggs or repagula encircling the twig beneath the egg-masses were reported (Henry,
1972, 78), no repagula were found in egg-masses of A. ramburi. The chorion is covered
with a reticulate pattern, and there is observed a micropylar area at each pole of the egg(Figs. I, 20). The chorion consists of two layers, that is, colored outer layer and transparent
inner, respectively. The inner layer is more resistive to sodium hypochlorite (antiformin)
than the outer layer.
There is a whitish gird ling line near the anterior pole of the egg (Figs. I, 2), and the first
instar larva hatches out from this part.

2.

Changes in the External Form of Developing Embryos

The egg period of A. ramburi is about 18 days at 19°C_28°C. The embryonic development of A. ramburi is divided into the following 16 stages.
Stage 1(about 1 day after oviposition, Fig. 2)

It is very likely that the maturation division and fertilization undergo early this stage, butthese processes could not observed in the present study.
The blastoderm formation follows as the cleavage nuclei i. e., the energids arrived at theegg surface late at this stage. The nuclei at the egg surface undergo the mitotic divisions repeatedly and the nuclear density increases, and then the cellular blastoderm is formed.

External morphogenesis of AscaZaphus embryo

205

MPA

:'.j''''';':'''::';''! :.: ....... :.. ~ ..:: ~ :,:.; .:: :,.:.:,.:.;' :.:\1...

1

2( .•..................................../ ...
:. """"~ .. "\ .. ~P"':"

MPL . .:""

3
1.
Fig. 2
Fig. 3Fig. 4Fig.

4Eggs of AscaZaphus ramburi.
Egg of Stage 1.
Egg of Stage 2, ventral view.
Egg of Stage 3, ventral view.

eN cleavage nucleus, GL girdling line,

LP lateral plate, MP A micropylar area, MPL
middle plate, PL protocephalon. Scales: 0.5 mm.

206Stage 2

Kamiya, A. and H. Ando(about 2 and 1/8 days after oviposition, Fig. 3)

The large heart-shaped ventral plate differentiates in the blastoderm. In the meanwhile
the rudimental middle and lateral plates begin to differentiates in the ventral plate. The segmentation of the ventral plate also begins at this stage.
Stage 3(about 2 and 1/4 days after oViposition, Fig. 4)

The germ band or ventral plate of this stage decreases in width, and its segmentation
proceeds. As the result of this, the protocephalon, intercalary, three gnathal, three thoracic
segments and an unsegmented abdominal lobe are formed. The middle and lateral plates
have differentiated but the inner layer has not yet formed.
Stage 4(about 2 and 3/8 days after oViposition, Fig. 5)

The germ band further decreases in width and the primitive groove along the middle lineof ventral plate begins to form. The protocephalon, intercalary, mandibular, maxillary,labial segments and the first to the third thoracic segments with a large abdominal lobe
become more apparent.
Stage 5(about 2 and 3/4 days after oviposition, Fig. 6)

The germ band becomes more elongated and width of the germ band becomes narrower.
The primitive groove also becomes clear, and the segmentation of the inner layer begins at
this time. The amniotic folds develop at the anterior and posterior ends of the germ band.
Stage 6(about 3 days after oviposition, Fig. 7)

The width of the germ band is much narrower than that in the former stage. The segmentation proceeds to the fourth abdominal segment whereas a large posterior part of theabdomen remains unsegmented.
Stage 7(about 3 and l/2 days after OViposition, Fig. 8)

The embryo or germ band continues to elongate further and its caudal end reaches near
the posterior egg pole. A pair of the labrum and antennal rudiments appear on the pro tocephalic lobes, and the rudiments of paired appendages are formed in each gnathal and thoracic segments except the intercalary. There are ten abdominal segments at this stage.
Therefore now the embryo consists of the protocephalic lobes, intercalary, three gnathal,
three thoracic and ten abdominal segments. The stomodaeal pit is seen just behind the labralrudiments.
Stage 8(about 4 days after oviposi~ion, Fig. 9)

The intercalary segment is gradually reduced, and a pair of appendages of the first abdominal segment becomes distingUishable at this stage. The appendages observed in the first ab-External morphogenesis of Asca/aphus embryo

207

AMF
PL

le

MDS
IL
MXS
LIS-r------..L
TS1.-----"
PG

AS1

5

6

\------tMX
1---------TLI

l--------tTL1

TS3-r-----1

7
Fig. 5.
Fig. 6
Fig. 7
Fig. 88

Egg of Stage 4, ventral view.
Egg of Stage 5, ventral view.
Egg of Stage 6, ventral view.
Egg of Stage 7, ventral view.

AMF· amniotic fold, ASl, 4,9, 1st, 4th, 9th abdominal segments, AT antenna, le intercalary segment, IL inner layer, LI labium, LIS labial segment, LR labrum, MD
mandible, MDS mandibular segment, MX maxilla, MXS maxillary segment, PG primitive groove, PL protocephalon, SD stomodaeum, TLI 1st thoracic leg, TSI-3 lst-3rd
thoracic segments. Scale 0.5 mm.

208Kamiya, A. and H. Andodominal segment are the pleuropodia, which are similar to the thoracic appendages in shape.
Stage 9(about 4 and 1/2 days after oviposition, Fig. 10)

The width of anterior part of the embryo increases, which may be seen especially fromthe protocephalic lobes to the thoracic region. A pair of the rudimental eyes appear on bothlateral sides of the protocephalic lobes, and the antennal rudiments elongate to the midventral line of the embryo. The stomodaeal pit is covered gradually by the developing
labrum. The rudiments of the mandible, maxilla and labium also develop, and the maxillarypalpal ru~iments are formed on each maxilla.
Stage 10(about 5 and 1/2 days after oviposition, Fig. 11)

The embryo becomes much wider than that in Stage 9. The stomodaeal opening is covered by the labrum, and the rudiments of eyes increase in size. The developing mandible andmaxilla elongate further, and the labium moves forwards and becomes to be located posteriorly to the maxilla. The rudimental appendages of the thorax and the pleuropodia develop
further. A pair of developing spiracles appear in the second and third thoracic segments andthe first to the eighth abdominal segments. The caudal end of the embryo bends to the ventral side and has the paired anal lobes.
Stage 11(about 6 and 1/2 days after oviposition, Fig 12)

The rudiments of lateral ocelli in the eye area are clearly observed in this stage. The
labium is hidden under the other gnathal appendages. It seems that the pleuropodia begin tosecrete. Brownish pigments appear over the surface of the head and both lateral sides of thethorax and abdomen of the embryo. The dorsal closure of the embryo begins, consequently
the width of the embryo increases further.
Stage 12(about 7 and 1/2 days after oviposition, Fig. 13)

The embryo develops so as to occupy the whole ventral side of the egg, and the dorsal
closure advances further. The basal parts of the mandible and maxilla begin to fuse, and apair of labial palpi appear at this stage. The scoli begin to develop on the second and third
thoracic segments. The pleuropodia are larger than those at the previous stage, and eachspiracle slightly rises from its original position. The tenth abdominal segment on which theanus is located reaches the six abdominal segment, and the ninth and tenth abdominal segments fuse with each other.
Stage 13(about 8 and 1/2 days after oviposition, Fig. 14)

The maxillae are located under the mandibles and the tips of these appendages attain to
the first abdominal segment. The developing eye composed of seven rudiments of lateralocelli changes into a thick and short process projecting from the head surface.
The thoracic legs elongate greatly, and the tips of metathoracic legs reach the seventh
abdominal segment. The basic pattern of the leg-segments is definitely formed. The tenthExternal morphogenesis of Ascalaphus embryo

209

LR
SO
AT
EYR
i-I---4-MO'M----+MX
f"±::I------+-LI.--.....,........

LI--r----+~'Y..-~+-----+TL3

TL 2-+-----j;~\~:M::::;=t----tPP

pp

9

10

LR
AT
EYR
MD
MX
LI

SP

TL2

11
Fig. 9Fig. 10.
Fig. 11
Fig. 12.

12

Egg of Stage 8, ventral view.
Egg of Stage 9, ventral view.
Egg of Stage 10, ventral view.
Egg of Stage 11, ventral view.

AL anal lobe, AS10 10th abdominal segment, AT antenna, EYR eye rudiment, LI labium,
LR labrum, MO mandible, MX maxilla, PO proctodaeum, PP p1europodium, SO
stomodaeum, SP spiracle, TL 1,2,3 1st, 2nd, 3rd thoracic legs. Scale: 0.5 mm.

210

Kamiya, A. and H. Ando

13

14

500
F~===::t:tPP.----:-==-~==_---"::tS

15
Fig. 13.
Fig. 14.
Fig. 15.
Fig. 16.

P

16

Egg of Stage 12, ventral view.
Egg of Stage 13, ventral view.
Embryo of Stage 13, ventral view.
Embryo of Stage 14, lateral view.

AT antenna, EY eye, LR labrum, MD mandible, MX maxilla, PD proctodaeum, PLP
labial palp, pp pleuropodium, SDO secondary dorsal organ, SP spiracle. Scale: 0.5 mm.

External morphogenesis of AscaZaphus embryo

211

abdominal segment is pointed-cone shaped.

Stage 14(about 10 days after oviposition, Figs. 15, 16)

The dorsal closure has already finished by this time and the large secondary dorsal organ
is formed on the dorsal side of the first thoracic to the third abdominal segments. The external form of the first instar larva has been completed except the scoli, tail and lack of thehairs.

Stage 15

(about 14 days after OViposition, Figs. 17, 18)

The eggs at this stage become dark in color and to be remarkably hollowed. The fullygrown embryo is almost similar to the first instar larva. The lateral ocelli turn dark brown,
the body is pigmented brownish, and the all bristles come out remarkably.

Stage 16(about 19 days after oviposition, Fig. 19)

The newly hatched larvae are brown, but they then change dark brown within a shorttime after hatching. They are a typical ascalaphid larva in many respects and about 5.2
mm in length including jaws. They stay on the egg-masses subsequent several days and thenfall on the earth.

Discussion
In general the form and size of the eggs of Ascalaphus ramburi resemble those describedfor other ascalaphids such as Hybris subjacens (Sonan, 1938), Ululodes mexicana, Ascaloptynx [urciger (Henry, 1972), and Byas albistigma (Henry, 1978). The structure and location of the micropyles observed in A. ramburi are basically the same as those in other species of Ascalaphidae. A. ramburi also shares with other owlflies a girdling line at the anterior pole of eggs for the hatching of larva, and the repagula are not formed.
In the egg of A. ramburi the large, broad ventral plate differentiates and this feature of
the ventral plate resembles those of Chrysopa perla (Bock, 1939) and Sialis mitsuhashii(Suzuki et aI., 1981), and moreover it bears also a resemblance to those of the scorpionfly,

Panorpa.
The segmentation of ectodermal part of the ventral plate in A. ramburi has occurred before the formation of the inner layer as reported in C. perla (Bock, 1939). Therefore thedifferentiation of middle and lateral plates and the segmentation in early developmental
stages seem to be the common features in the neuropteran embryogenesis.
The embryo of A. ramburi develops on the egg surface as observed in S. lutaria (Strindberg, 1951; Du BOis, 1938), C. perla (Bock, 1939) and Protohermes grandis (Miyakawa,1979). The caudal end of the embryos in S. lutaria, C. perla and P. grandis reaches the dorsal side of the egg, and the katatrepsis of the embryo is remarkable, but the embryonic caudal end of A. ramburi attains to only the posterior pole of the egg because of the shortnessof embryo, and conspicuous katatrepsis does not occur. Seven lateral ocelli develop in A.
ramburi, whereas six in S. lutaria, C. perla and P. gandis.

212

Kamiya, A. and H. Ando,\Hccff-:--;f.-;*PL P
pp

18

17

se

Fig. 17.
Fig. 18.
Fig. 19.
Fig. 20.

Embryo of Stage 15, ventral view.
Embryo of Stage 15, lateral view.
1st instar larvae (Stage 16), dorsal view.
Micropylar area at anterior pole of egg (arrow, micropyle) (X360)

AT antenna, EY eye, MD mandible, MP micropyle, MX maxilla, PLP labial palp,
PP remnant of pleuropodium, se scolus, SP spiracle. Scales: 50 Jlm in Fig. 20, 0.5 mm
in others.

External morphogenesis of Ascalaphus em bryo

213

General features of external morphogenetic process observed in A. ramburi may allow us
to consider that they belong to those commonly found in the primitive holometabolous
insects.

Acknowledgement
We thank to Mr. Tachu Koshimizu for his help to the present study. A scanning electron
micrograph of Asca/aphus egg used in this paper is indebted to Prof. Morio Matsuzaki ofBiological Laboratory, Fukushima University.

References
Bock, E. 1939. Buildung und Differenzierung der Keimbllitter bei Chrysopa perla (L.).
Z. Morphol. Okol. Tiere. 35: 615-702.
Bock, E. 1941. Wechselbeziehungen zwischen den Keimbliittern bei der Organbildung vonChrysopa perla (L.). I. Die Entwicklung des Ektoderms in mesodermdefekten Keimteilen. Wilhelm Roux's Arch. 141: 159-247.
Du Bois, A. M. 1936. Recherches experimentales sur la determination de l'embryon dansl'op.uf de Sialis lutaria. Rev. Suisse Zool. 43: 519-523.
Du BOis, A. M. 1938. La determination de l'ebauche embryonaire chez Sialis lutaria L.
(Megaloptera). Rev. Suisse Zool. 45: 1-92.
Henry, C. S. 1972. Eggs and repagula of Ululodes and Ascaloptynx (Neuroptera: Ascalaphidae): a comparative study. Psyche, 79: 1-22.
Henry, C. S. 1978. The egg, repagulum, and larva of Byas (Neuroptera: Ascalaphidae):morphology, behavior and phylogenetic significance. Syst. Entmol. 3: 19-34.
Miyakawa, K. 1979. The embryology of the dobsonfly, Protohermes grandis Thunberg(Megaloptera: Corydalidae). 1. Changes in external of the embryo during development.
KontyCt, 47: 367-375.
Miyakawa, K. 1980. Embryogenesis of the pleuropodia and abdominal filament (tracheal
gills) in Protohermes grandis Thunberg (Megaloptera: Corydalidae). Abstract of 16th
Int. Congr. Entomol. (Kyoto), 1980: 50.
Okamoto, H. 1909. On Japanese Ascalaphidae. Zool. Mag. 21: 499-508 (in Japanese).
Sonan, J. 1938. On the life-history of Hybris subjacens Walker (Neuroptera: Ascalaphidae). Trans. Nat. Hist. Soc. Formosa, 28: 272-274 (in Japanese).
Strindberg, H. 1915. HauptzUge der Entwicklungsgeschichte von Sialis lutaria L. Zool.
Anz., 46: 167-185.
Suzuki, N., S. Shimizu and H. Ando 1981. Early embryology of the alderfly, Sialis mitsuhashii Okamoto (Megaloptera: Sialidae). Int. J. Insect Morphol. Embryol. 10: 409418.
Authors' address

Mr. A. Kamiya,
Senior High School attached to Aichi
Kyoiku University,
Kariya, Aichi 449, Japan
Prof. H. Ando,
Sugadaira Montane Research Center,
University of Tsukuba,
Sanada, Nagano 386-22, Japan