Recent Advances in Insect Embryology in Japan and Poland
Edited by H. Ando and Cz. Jura
Arthropod. EmbryoL. Soc. Jpn.
(ISEBU Co. Ltd., Tsukuba) 1987

225

Embryonic Development of Nervous Systemin the Alderfly, Sialis mitsuhashii Okamoto(Megaloptera, Sialidae) *Nobuo SUZUKI
Satoru SHIMIZU
andHiroshi ANDO
Synopsis
The nervous system formation in the embryo of the alderfly, Sialis mitsuhashii Okamoto(Megaloptera, Sialidae), is described. The ventral nerve cord is formed mainly from neuroblasts
occurred in three gnathal, three thoracic, and ten abdominal segments. The median cord seems
to contribute the formation of the transverse commissure and dorsal cortical layer of the ganglion. The modified outer ganglion cells give rise to the perilemma. The embryogenesis of thebrain occurs in usual manner as in most insects. The stomatogastric nervous system is derived
from three evaginations along the medio-dorsal part of the stomodaeal roof. The anterior evagination provides the frontal ganglion, and the middle and posterior ones the hypocerebral ganglion and small ganglion-like swelling, which degenerates up to hatching, respectively.

Introduction
The Megaloptera has been considered as the most primitive order of holometabolous insects because of its fossil record, and the embryology of this order, which consists of two families, Sialidae and Corydalidae, is poorly known.
As far as the authors are aware, there are only six papers, concerning Sialis (Siali-* Contributions from Sugadaira Montane Research Center, University of Tsukuba, No. 113.

226

N. Suzuki, S. Shimizu and H. Andodae): Strindberg (1915) - general embryology, Du Bois (1938) - experimental research with brief description of normal embryology, Suzuki et al. (1981) - early
embryogenesis, Ando et al. (1985) - external morphology of embryogenesis; and Protohermes (Corydalidae), Miyakawa (1979) - external features of embryos through development, Miyakawa (1980) - embryogenesis of abdominal appendages.
As mentioned above, the embryology of the Megaloptera is known only briefly,
espcially as for the organogenesis. Accordingly, this paper deals with the formation of
the nervous system, and this study might furnish data useful in understanding the relationship between the Megaloptera and other lower holometabolans.

Materials and Methods
Pregnant females and eggs of Sialis mitsuhashii Okamoto were captured at theGoshiki Lake, Fukushima Prefecture of Japan in mid-June, 1978-1982. Fixation andsectioning of the eggs were done in same manner as described in the previous paper(Suzuki et al., 1981).

Observationsa) Ventral nerve cord
At 55 hours after oviposition, two to three neuroblasts differentiate in the eachlateral plate of gnathal, thoracic, and first and second abdominal segments (in crossand longitudinal sections), though there is no neuroblast in the developing median cordyet.
At 60 hours after oviposition, mesodermal cells on the lateral plates become a thinsingle layer, and the number of neuroblasts contained in these plates increases to four
to five in longitudinal section (two to three in cross section). The median cord of theinter- and intrasegmental regions in cross section is rather broad at this stage. The
posterior region of the median cord has a cell, stained lightly with hematoxylin, andhas a large nucleus in the medio-dorsal portion (Fig. I), and this may correspond to aneuroblast of the lateral plate.
At about 70 hours after oviposition, the neuroblasts appear in the remaining segments, i. e., to the tenth abdominal segment. The number of neuroblasts at lateralplate of each segment becomes to three to four in number in cross section and five to
six in longitudinal section, however four to five in longitudinal section of the tenthabdominal segment. The neuroblasts repeat mitoses to produce their daughter cells
above them, so that the columns of nerve cells are formed above these neuroblasts (Fig.
2). As development proceeds the daughter cells, at dorsal portion of the column, start
to form the neuropile (Fig. 3). There is a thin mesodermal cell layer on the neuropile,
however this layer never differentiates into the perilemma. As ganglia increase in size,
the medio-ventral part of the intersegmental median cord becomes narrow in cross section (Fig. 3). On the other hand, the cord of the posterior part of each segment and intrasegmental region still possesses its original feature, and contains a neuroblast in its

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Formation of nervous system in Sialis mitsuhashii

nbm

4
mdc

np

mdc

3

np

pe

nbm

pe

6

7

Figs. 1-7. Embryonic development of ventral nerve cord in Sialis mitsuhashii.
I. Transverse section through posterior part of labium in a 60-hr embryo. 2. Parasagittal section through maxilla to first thorax in a 70-hr embryo. 3 - 6. Transverse sections through second thorax in a 72-hr embryo (Fig. 3), maxilla in a 90-hr
embryo (Fig. 4), second thorax in a 4-day embryo (Fig. 5), and first thorax in a 5day embryo (Fig. 6). 7. Sagittal section through first thorax in a 6-day embryo.
dnb, daughter cell of neuroblast; dpe, developing perilemma; lis, labial segment; mc,
mesodermal cell; mdc, median cord; mxs, maxillary segment; nb, neuroblast; nbm, neuroblast of median cord; np, neuropile; pe, perilemma; tl, first thoracic segment; tl2 appendage
of second thoracic segment; y, yolk. Scale: 50 f.L m.

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N. Suzuki, S. Shimizu and H. Andomedio-dorsal part.
At 80 hours after oviposition, the mesodermal layer almost disappears above thedeveloping ganglia, and the ganglia increase in height as the neuroblasts repeat mitoses
and produce nerve or ganglion cells. The neuropile at the dorsal part of each ganglion
becomes apparent, though there are observed no intrasegmental connectives. The
peripheral ganglion cells begin to be slightly flat and cover the developing ganglia. As
development advances those flattened cells become membranous and seem to be a
rudimental perilemma (Fig. 4). As a result of the development of ganglia, the ventral
half of the intersegmental median cord of each segment becomes thin in cross section,
though it connects to the epidermis. The dorsal half of the intersegmental median cordtakes a feature of inverted triangle in cross section, and becomes to be light-stained, sothat it becomes difficult to discriminate from the developing neuropile of the ganglia(Fig. 4).
At 4 days after oviposition, the neuropile formation further undergoes, and theanterior and posterior commissures, though only one commissure in mandibular ganglion, and connectives of each ganglion are observed. Between both the commissures,the medio-dorsal region of the median cord seems to participate in the formation of thedorsal cortical layer of the ganglion (Fig. 5). There are a few mitotical figures of theneuroblasts in the ganglia now increased in height, and right and left ganglia begin to
move towards the median line. Consequently, the median cord is almost detached fromthe epidermis throughout the intersegmental regions, on the other hand, they possess
the connection with the epidermis at the intrasegmental regions. The ganglia of thethree gnathal segments begin to gather.
At 5 days after oviposition, the connectives are formed between each ganglion andcovered with the perilemma, and it nearly covers the dorsal part of the each ganglion
also (Fig. 6). From lateral sides of the gnathal and thoracic ganglia, a pair of neurofiber differentiates and extends into each appendage. The median cord finally loses theconnection at the intersegmental regions, and is nearly detached at the intrasegmental
regions. The gnathal ganglia further fuse, and become a rudimental suboesophageal
ganglion.
At 6 days after oviposition, the embryo begins to revolve, and the perilemma completely covers the dorsal part of each ganglion. The eighth to tenth abdominal ganglia
begin to gather and fuse. The cell mass of the intrasegmental median cord, which contains a neuroblast, exists at the medio-dorsal region of the posterior part of each ganglion (Fig. 7).
At 7 days after oviposition, the eighth to tenth abdominal ganglia become morecompact, and situate just behind the seventh abdominal ganglion. The neuroblast of
the median cord, located in the posterior margin of each ganglion, becomes undetectable from ganglion cells. As development advances, the epidermis begins to be thin, sothat the ganglia are almost removed from the epidermis.
At about 9 days after oviposition, the ganglia are finally detached from theepidermis, and they approximately get the same features of those of the first ins tar larvae.

Formation of nervous system in Sialis mitsuhashii

229

11

13

10
sup

19

11
Figs. 8 - 11. Embryonic development of brain in Sialis mitsuhashii. Horizontal sections
through head.
8. 55-hr embryo. 9. 70-hr embryo. 10. 4-day embryo. 11. 5-day embryo.
dc, deutocerebrum; fg, frontal ganglion; ill, invagination of lobus I; 11 - 3, lobi
- 3 of protocerebrum; mc, mesodermal cell; nb, neuroblast; sd, stomodaeum; sup,supraoesophageal commissure; t, tentorium; y, yolk. Scale: 50 pm.

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N. Suzuki, S. Shimizu and H. Andob) Brain

The ectodermal cells of protocephalon, antennal and intercalary segments areroughly uniform in size by 2 days. However, there arise several cells containing large
nuclei in the ectoderm of those segments at 55 hours after oviposition. They areneuroblasts, and as development undergoes their nuclei become to be faintly stainedwith hematoxylin. The neuroblasts at the protocephalic lobes are distributed into threegroups, which are corresponding to the future lobi I - 3 of the protocerebrum, on eachlobe (Fig. 8). The neuroblasts of the antennal segment, which form the deutocerebrum
later, are observed at the both sides of the developing stomodaeum, namely at the basal part of antennal anlagen, and those of the intercalary segment produce the future
tritocerebrum, and are found just behind the neuroblasts of the antennal segment.
The mitoses of neuroblasts occur, and at 70 hours after oviposition the neuroblasts
frequently repeat teloblastical divisions. At the ectoderm of future lobus I of the protocerebrum, the teloblastical mitoses are not observed, but found the invagination of
the ectoderm containing the neuroblasts (Fig. 9), so that the rudimentallobus I seems
to be derived from the invaginated ectoderm at first. At this stage the protocerebral
lobi 1 - 3 become easily recognizable, and piled daughter cells of the lobi 2 and 3 start
to differentiate into the neuropiles at their dorsal regions. The neuropile of the brain isformed in the same way as that of the ventral nerve cord.
At 3 days after oviposition, developing deuto- and tritocerebra slightly shift forward, and the deutocerebrum locates behind the protocerebral lobus 3. The appearance of the neurilemma occurs, and its formation is similar to that of the ventral nerve
cord. At 4 days, the distal region of the lobus 1 begins to be detached from theepidermis (Fig. lO). The supraoesophageal commissure makes its appearance between
the lobi 3, and the neuropile is discernible in the commissure (Fig. lO). The neuropile
between the lobi 2 and 3 is also visible, and the lobi I - 3 are removed from theepidermis. The deutocerebrum moves forward, and the tritocerebrum takes a position
just behind the former. As development proceeds, the three cerebra connect with eachother by each connective, and there occur the connectives between the tritocerebrum
and mandibular ganglion also.
At 5 days after oviposition, the protocerebral lobi I and 2 shift forward and lobus
3 shifts medially. The distal end of the lobus I connects with the epidermis again, andthis distal part and epidermis connecting with the lobus I later become the rudimental
postretinal fiber and optic plate respectively. The commissure of the deutocerebrum,locating beneath the protocerebrum, becomes confluent with the protocerebral commissure, and it seems to form the supraoesophageal commissure (Fig. 11). There appears a
tritocerebral commissure, i. e., suboesophageal commissure under the stomodaeum.
The tritocerebrum sends neurofibers to the frontal ganglion. The protocerebral lobi 13 shift mediad further, and the neuropilar region develops in them, though it is illdeveloped in the lobus I, at 6 days after oviposition. The lobus I is divided into distaland proximal parts, and the epidermis connected with the distal end is slightly thickened, so that the optic plate begins to differentiate. The deutocerebral nerves innervate
the antennae.
At 7 days, the protocerebrum takes a position horizontal above the stomodaeum inthe embryonic head capsule. The neuropiles further develop among the cerebra, andFormation of nervous system in Sialis mitsuhashii

231

also in the protocerebral lobus 1 as development advances. At 8 days after oviposition,
the three cerebra consolidate into a single mass, viz., brain. The brain assumes an
ellipsoid shape and lies horizontally over the stomodaeum. In the cortical layer at eachposterior margin of the protocerebrum, two large neurosecretory cells are constantly
observed. No significant changes are found in the brain development up to the time ofhatching.
c) Stomatogastric nervous .rystem

At 60 hours after oviposition, there arise three pits along the median line of thedorsal wall of the developing stomodaeum. These pits later become three evaginations,by which the stomatogastric nervous system is produced. The ectoderm which lies on
the pits has a few large cells (Fig. 12). At 70 hours, there occur three small evaginations along the median line of the stomodaeal roof (Fig. 13) and they are covered withmesodermal cells. At 3 days after oviposition, the evaginations change into three cellmasses and begin to extend forward. At 5 days after oviposition, the anterior cell massfrom the anterior evagination produces the anlage of frontal ganglion (Fig. 14). Simultaneously the anterior and middle cell masses are detached from the stomodaeal roof.
As for the posterior mass, it has still a connection with the roof.
At 6 days after oviposition, the rudimental frontal ganglion migrates forward andlocates in front of the supraoesophageal commissure. There appear the nervefibers between the frontal ganglion and tritocerebrum, and are recognizable the neuropilar regions in these ganglia. The middle cell mass becomes corded and forms a recurrent
nerve running from the frontal ganglion to the osesophagus (Fig. 15), and then thecord gets a small thickening, which is a future hypocerebral ganglion and corresponds
to the oesophageal ganglion in Sialis lutaria (Strindberg, 1915), at its middle portion.
The posterior mass exists on the stomodaeal roof near its blind end, and consists oflight-staining cells. The anterior part of the posterior mass extends forward and connects with the developing recurrent nerve. At 7 days after oviposition, the posterior cellmass finally begins to be detached from the stomodaeal roof. There appears the neuropile within the middle swelling of the cord. At 8 days, the frontal ganglion further migrates forward, and the posterior cell mass produces a ganglion-like swelling. The
neuropile differentiates within the swelling, which situates just behind the anlage of
hypocerebral ganglion (Fig. 16). This swelling becomes ill-developed up to the time ofhatching.

Discussion
Median cord
As for the fate of the median cord, Baden (1936) concluded that it does not participate in the ganglion formation in orthopteran Melanoplus. According to generalagreement about role of the cord, however, it shows a contribution to the ganglion
formation in many insects.

N. Suzuki, S. Shimizu and H. Ando

232

Ir

ps

fg

s

Ir

15
hg

16

fg

Figs. 12-16. Embryonic development of stomatogastric nervous system in Sialis mitsuhashii.
12. Slightly oblique sagittal section through head of a 60-hr embryo. 13. Transverse section through head of a 70-hr embryo.
14. Slightly oblique sagittal section through head of a 5-day embryo. Sagittal section through head of a 6-day
embryo. 16. Sagittal section through stomatogastric nervous system of a 8-day
embryo.
fg, frontal ganglion; hg, hypocerebral ganglion; lr, labrum; mc, mesodermal cell; np, neuropile; ps, pit of stomodaeal roof; rn, recurrent nerve; sd, stomodaeum; sr, stomodaeal roof; y,yolk. Scale: 50 f.l m.

Formation of nervous system in Sialis mitsuhashii

233

For example, the median cord in part forms the transverse commissure andperilemma of ganglion in lepidopteran Chilo (Okada, 1960) and Endoclita (Tanaka et al.,1985). The cord produces glial elements in hemipterans (Springer and Rutschky, 1969)
and coleopteran Lytta (Rempel et al., 1977). On the other hand, it participates in bothformations of the glial element and dorsal cortical layer of the ganglion in trichopteran
Stenopsyche (Miyakawa, 1974) and lepidopteran Neomicropteryx (Kobayashi and Ando,
1983).
In Sialis, the median cord principally contributes to the formation of the transverse
commissure and the dorsal cortical layer of the ganglion, though there is a little possibility that the glial element partially develops from the cord. Consequently the role ofthe median cord in Sialis is thought to be similar to that of Endoclita (Tanaka et al.,1985).

Brain
Miyakawa (1974) and Kobayashi and Ando (1983) reported that the lobus 1 ofthe protocerebrum is made from daughter cells of the neuroblasts. In Sialis, the lobus 1
occurs from the invagination of the ectoderm containing neuroblasts at embryonic headlobe. The neuroblasts, however, seem not to divide teloblastically and may correspondto the neuroblast-like cells which appear in the course of development of the stomatogastric nervous system in coleopteran Tenebrio (Ullmann, 1967), so that they may not
be true neuroblasts in the strict sense. The fact that the lobus 1 occurs from theectodermal invagination agree with the view of Ullmann (1967) and Rempel et al.
( 1977).
Although the deutocerebra1 commissure is absent in some insects (Eastham, 1930;
Tanaka et al., 1985; etc. ), in Sialis the proto- and probably deutocerebral commissuresform the supraoesophageal commissure as in Tenebrio (Ullmann, 1967) and Stenopsyche(Miyakawa, 1974), and the suboesophageal one occurs in usual manner observed inmany insects.

Perilemma
The perilemma consists of a cellular uni-layer, the perineurium, and its secretion
product represents the neurilemma (Rempel et al., 1977). Though there are several
opinions in regard to origin of the perilemma in ventral ganglion, they agree in point of
the fact that it derives from the ectoderm, except for Baden's view (1936).
Okada (1960) concluded that the peri1emma originates from the median cord, andmore generally it is thought that it occurs from modified outer ganglion cells (Nelson,
1915; Miyakawa, 1974; Rempel et al., 1977; Kobayashi and Ando, 1983; and in Sialis).
Besides, according to Ullmann (1967) and Tanaka et al. (1985) the perilemma has adual origin, namely formed from the modified ganglion cells and median cord. Theremay be no phylogenetic significance as for the origin of the perilemma as same as thefate of the median cord, judging from the facts mentioned above. However the ultras-

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N. Suzuki, S. Shimizu and H. Andotructural investigations should be demanded promptly for those embryogenesis in detail.

Stomatogastric nervous system
The stomatogastric nervous system is derived from cell masses originated from thestomodaeal ectoderm along its dorso-median line. The number of the cell mass is one
in Chilo (Okada, 1960), two in Epiophlebia (Ando, 1962) and Pieris (Eastham, 1930),
and three in most insects (Anderson, 1972). Even in the last case, there are various
opinions about the products of those cell masses except for the anterior cell mass,
which gives rise to the frontal ganglion.
In Sialis, the middle cell mass provides the recurrent nerve and hypo cerebral ganglion as in the case of Ullmann (1967), Rempel et al. (1977) and Kobayashi and Ando
(1983), and the ganglion-like swelling occurred from the posterior mass seems to correspond with the ventricular ganglion, mentioned by those authors.

References
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Du Bois, A. M., 1938. La determination de l'evauche embryonnaire chez Sialis lutaria L. (Megaloptera). Rev. Suisse Zool. 45: 1-92.
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Authors' addresses: Dr. N. Suzuki
Japan Women's College of Physical
Education, Kitakarasuyama, Setagaya,
Tokyo 157, JapanMr. S. Shimizu and Pro( H. Ando
Sugadaira Montane Research Center,
University of Tsukuba, Sanada, Nagano 386-22, Japan