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

215

Embryonic Development of the Ventral Nervous Systemof the Stonefly, Kamimuria tibialis (Pictet)(Plecoptera, Perlidae) *Tohru KISHIMOTO
Synopsis
The embryonic development of ventral nervous system in a plecopteran Kamimuria tibialis
was described, with special reference to the change in number of neuroblasts in the gnathothoracic to the abdominal segments during embryogenesis.

Introduction
I have been studying the embryonic development of the stonefly, Kamimuria tibialis
to understand the basic developmental pattern of the Plecoptera, and to examine thephylogenetic relationship between the Plecoptera and other polyneopteran orders fromthe embryological point of view.
In our previous papers on the embryogenesis of K. tibialis (Kishimoto and Ando,
1985, 1986), we described the changes in the external features of the embryo and theformation of the alimentary canal, but the embryogenesis of other organs remains unpublished.
In the present paper, I describe the embryogenesis of the ventral nervous system
of K. tibialis.

Materials and Methods
Most of the information concerning materials and methods utilized in the present* Comprising a part of the doctoral thesis, University of Tsukuba, 1986 (unpublished).
Contributions from Sugadaira Montane Research Center, University of Tsukuba, No. 112.

216

T. Kishimoto

study were described in a previous paper (Kishimoto and Ando, 1985).
For sectioning, normal paraffin embedding method was employed. Sections, 6 p. mthick, were stained with Delafield's haematoxylin and eosin.

Results
Embryonic development in K. tibialis divided into 12 stages, based chiefly onchanges in external features of the embryo. Stage numbers used in the present study
correspond to those in the previous paper (Kishimoto and Ando, 1985).

a) General development of ventral nerve cord
Formation of the ventral nerve cord is shown diagrammatically in Fig. 1.
In stage 4, a slight median groove is formed at the ventral surface of the embryo.
This groove is said to be a rudiment of neural groove and indicates the sign of the beginning of the neurogenesis, but the neuroblasts could not be found at this time. In
stage 5, the gnathal, thoracic and first four abdominal segments of the embryo areformed and several large cells or neuroblasts differentiate among the ectodermal cells ineach of the gnathal and thoracic segments. The ectodermal layer on the neural groove
invaginates to form the median cord at the dorso-median line of the embryo, but theneuroblasts of the median cord do not differentiate yet. The portion of median cord in
the anterior region of each segment becomes thinner than that in the posterior region.
In stage 6, at the time the ectodermal segmentation of the embryo completes tothe II th abdominal segment, the neuroblasts become distinct in the intercalary to thesecond abdominal segment (Fig. I A). They are easily distinguished from the surrounding ectodermal cells, since their nuclei are large (ca. 10 p. m in diameter) andstained lightly with haematoxylin and the neuroblasts produce small daughter cells
which are called as ganglion mother cells by their unequal divisions. The neuroblasts
at each lateral plate in a segment are four or five in number in cross and longitudinal
sections.
The differentiation of the neuroblasts is delayed in the posterior segments. Early instage 7, the neuroblasts make their appearance in the four gnathal, three thoracic and
10 abdominal segments. In the gnathal and thoracic segments, the production of thedaughter cells or ganglion cells proceeds, so that the columns of the ganglion cells areformed.
As the same time the ganglion cells of the gnathal and thoracic segments form
plasmic fibers which in turn form the bundles of the axon or neuropiles, situated independently of each other (Fig. 1 B). As the development proceeds, the neuropiles increase in volume and then extend antero-posteriorly. Late in stage 7, the ganglia of theintercalary to the first abdominal segment are connected longitudinally with the extending neuropiles or connectives (Fig. I C).
In this stage a neuroblast differentiates in the posteriormost part of each mediancord of the mandibular to the metathoracic segment.
In stage 8, the neuroblasts also appear in the II th abdominal segment, but they
are only two or three in number in the lateral half of this segment (Fig. 1 D). They doFormation of ventral nervous system in Kamimuria

217

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Fig. 1. Schema of the formation of ventral nervous system. A, B, C, D, E, F; stage 6, earlyin stage 7, late in stage 7, stage 8, stage 9 and stage 12, respectively. Arrows showing the anterior shifting of ganglia. AS 1- 11, first to II th abdominal segment; Cm,
commissure; Cn, connective; GAS7-1O, ganglia of seventh to 10th abdominal segment; GTh3-GASI, ganglia of metathoracic and first abdominal segments; InS, intercalary segment; Li, labial segment; Md, mandibular segment; Mx, maxillary segment; Nb, neuroblast; NbMC, neuroblast of median cord; Np, neuropile; SG, suboesophageal ganglion; ThSI-3, pro-, meso- and metathoracic segments.

T. Kishimoto218

not produce the daughter cells and disappear in the next stage. Consequently, theganglion of the 11 th abdominal segment is not formed. The neuroblast of the mediancord in this segment also does not differentiate.
The columns of the ganglion cells of each segment grow higher, especially in thegnathal and thoracic segments, and each ganglion increases in volume, while the dermatoblasts become attenuated and begin to form the epidermis. The ganglion cells,
produced by the neuroblasts at the dorso-lateral part of each segment, do not form thecolumn, but extend medially on the dorsal surface of the ganglia to form the coveringof the ganglia. In the median cord of the second to the fourth abdominal segment, theneuroblasts differentiate.
The neuropiles appear in the intercalary to the ninth abdominal segment and increase in volume. The connectives are formed between each segment from the intercalary to the fourth abdominal segment and become thicker than in the previous stage. In
this stage the transverse connections of the neuropiles of each ganglion, so-called commissures, are formed in the mandibular to the fourth abdominal segment. Two commissures are formed: the anterior and posterior, in each segment but only one in themandibular segment.
In stage 9 the dermatoblasts become thinner and are separated from the ganglion. The neuroblasts of the median cord occur from the mandibular to the 10th abdominal segment (Fig. 1 E). The connectives and commissures are completed in thegnathal, thoracic and abdominal segments; two commissures in each segment except in
the intercalary, mandibular and 10th abdominal segments, where only one commissure
is formed. The neuropiles increase in volume and occupy the most part of the ganglion
in each segment.
The neurilemma makes its appearance on the dorsal surface of the ganglion as athin membrane. The origin of the neurilemma is unclear, but it may be originated fromthe peripheral cells covering the ganglion, produced by the dorso-lateral neuroblasts.
From this stage the fusion of the ganglia begins in the three regions. With the
movements of the cephalization, the mandibular, maxillary and labial ganglia shift forwards to fuse with each other, while the ganglia of the intercalary segment also shift
forwards and come to lie laterads of the stomodaeum. The first abdominal ganglion begins to shift to the metathoracic ganglion, and the eighth to the 10th abdominal ganglion begin to shift to the seventh abdominal ganglion. As the development proceeds,
the sternum of the first abdominal segment fuses with the metathoracic sternum, but
the seventh to the 10th abdominal segment do not fuse with each other.
In stage 12, in the full-grown embryo, the fusion of the ganglia in the three regions
completes; the gnathal ganglia fuse to form the suboesophageal ganglion, the seventh tothe 10th abdominal ganglion fuse to form the large ganglion (Fig. 1 F). All the neuroblasts in each segment disappear since the neurblasts differentiate into the ganglion
cells. The commissures in each segment become thickened, so that the anterior andposterior ones cannot be distinguised from one another.

b) Number of neuroblasts in each segment
During the embryonic development of the ventral nervous system the whole num-Formation of ventral nervous system in Kamimuria219

ber of neuroblasts in each ganglion of the mandibular to the 10th abdominal segment
was counted. The cell counts were made on the reconstructions of serial sections, andportions of the one neuroblast could be traced for two or three sections because of its
large diameter (ca. 10 ,u m).
Number of neuroblasts in gnathal segments:
The neuroblasts of each gnathal segment (mandibular, maxillary and labial segments) appear in stage 6. They increase in number and reach the maximum number instage 7: the maximum number of neuroblasts is 42 in the maxillary and labial segments, and 28 in the mandibular segment. The number of neuroblasts in each gnathal
segment begins to decrease after this stage and by stage II all the neuroblasts degenerate. The time course of change in the number of neuroblasts is similar within the maxillary and labial segments.
Number of neuroblasts in thoracic segments:
The neuroblasts of each thoracic segment appear in stage 6. They increase in
number and reach the maximum number in stage 7: the maximum number of neuroblasts in 67 in the prothorax, 65 in the mesothorax and 60 in the metathorax. The number of neuroblasts in each thoracic segment begins to decrease after this stage and then
all the neuroblasts degenerate late in stage 11. The time course of change in number of
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Fig. 2. Change in number of neuroblasts in time course. The mean number of neuroblasts
per segment was calculated for the three thoracic and two gnathal (maxillary andlabial) segments, and plotted against embryonic age. Arrows showing the differentiating time of neuropile (Np), connective (Cn), commissure (Cm) andneuroblast of median cord (NbMC).

T. Kishimoto220

neuroblasts among three segments is quite similar to each other.
Number of neuroblasts in abdominal segments:
The neuroblasts of the first and second abdominal segments appear at the beginning of stage 6, and those of the posterior segments appear late in stage 6 or stage 7.
The maximum number of neuroblasts in each abdominal segment is: 43 in the firstabdominal segment, 42 in second, 39 in third, 36 in fourth, 32 in fifth, 34 in sixth, 39
in seventh, 34 in eighth, 28 in ninth and 10 in 10th. Apparently, the number ofneuroblasts in the 10th abdominal segment is smaller than that of other segments.
Comparison of the time course change in the number of neuroblasts among gnathal,
thoracic and abdominal segments:
The time course change in the number of neuroblasts in gnathal, thoracic andabdominal segments is shown in Figs. 2 and 3.
In Fig. 2, the mean number of neuroblasts per segment is plotted at the various
stages for the three thoracic segments, and the maxillary and/or labial segments. This
figure shows that the neuroblasts in the gnathal segments degenerate more rapidly thanin the thoracic segments.
In Fig. 3, the mean number of neuroblasts per segment is plotted for the three3

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Fig. 3. Change in number of neuroblasts in time course. The mean number of neuroblasts
per segment was calculated for the three thoracic and first three abdominal segments, and plotted against embryonic age, also the number of neuroblasts in the
ninth and 10th abdominal segments was plotted. Arrows showing the differentiation
time of neuropile (Np), connective (Cn), commissure (Cm) and neuroblast of median cord (NbMC).

Formation of ventral nervous system in Kamimuria

221

thoracic segments and first three abdominal segments, additionally, the number ofneuroblasts in the ninth and the 10th abdominal segments is also plotted. This figure
shows that the neuroblasts of the abdominal segments degenerate most rapidly in thethree major regions of the embryo, and that the segmental specificity occurs within theabdominal segments in early neurogenesis.

Discussion
The general development of the ventral nervous system in K. tibialis proceeds inthe same way as in other insects. In K. tibialis the neuroblasts differentiate in threegnathal, three thoracic and 11 abdominal segments, but those in the 11 th abdominal
segment are transient and do not form a pair of abdominal ganglia. Also Allonarcys ( =Pteronarcys) proteus, the 11th abdominal ganglion is not formed (Miller, 1940). According
to Johannsen and Butt (1941), during the earlier stages of development, in more primitive insects as well as in some of more advanced ones (Lepisma, Gryllotalpa, Periplaneta,
Gryllus, Locusta, Leptinotarsa, Donacia, Calandra, Hylotoma, Charicodoma, Apis,and others),
the 11 th abdominal ganglion can be identified but they disappear as the development
proceeds.
In the later development of insects, there is a tendency for some of the ganglia,
usually the eighth to the 10th (11th) abdominal one, to fuse. Further fusion of theganglia may also occur, depending on the orders of insects, especially in the shortbodied paraneopterans.
In K. tibialis the seventh to the 10th abdominal ganglion fuse and, the metathoracic and the first abdominal ganglia do so also, while in A. proteus, only the eighth tothe 10th abdominal ganglion fuse (Miller, 1940).
The median cord of K. tibialis is derived from the median invagination of the ventral ectoderm, in the same way as in other insects, but the fate of it could not be
traced. According to the detail work of Goodman and Spitz er (1979), each neuroblast of
the median cord gives rise to the dorsal median neuron.
Concerning the origin of the neurilemma in the ventral ganglia, there are threedifferent opinions: 1) the neurilemma originates from the peripheral ganglion cells(Heymons, 1895; Strindberg, 1913; Nelson, 1915; Eastham, 1930; Roonwal, 1937;
Ando, 1962), 2) from the median cord (Nusbaum, 1883; Wheeler, 1893; Tiegs andMurray, 1938; Okada, 1960), 3) or is mesodermal in origin (Baden, 1936). In K.
tibialis, the neurilemma seems to arise from the peripheral ganglion cells.
The number and arrangement of neuroblasts were investigated in the orthopteran
Locusta migratoria (Bate, 1976) and Gryllus bimaculatus (Miyamoto and Shimozawa, 1983;
Miyamoto, 1983), and in the phasmid Carausius morosus (Tamarelle et al., 1985)
embryos.
Bate (1976) investigated the formation process of thoracic and abdominal ganglia
during the embryogenesis of L. migratoria. He confirmed that the arrangement andnumber of neuroblasts are very regular and segmentally homologous: neuroblasts ineach of three thoracic segments are arranged in seven rows composed of four to 10 cells
plus one median neuroblast, with 60 + 1 cells in each segment. In the abdomen thearrangement of neuroblasts is similar to that of thorax, with 56 + 1 cells per segment.

222

T. Kishimoto

According to Miyamoto and Shimozawa (1983) and Miyamoto (1983), the number and arrangement of neuroblasts for all segments resemble each other. There are 62+ 1 neuroblasts in each of the gnathal and thoracic segments, and 56 + 1 in theabdominal ones. In C. morosus, the map of thoracic neuroblasts shows the presence of
56 elements in each thoracic segment (Tamarelle et al., 1985).
Thus, in the early embryogenesis the anlage of the nervous system shows the segmental homology, not only within the thoracic and the abdominal ganglia, but also between the thoracic and abdominal ones.
In K. tibialis, the segmental homology within the thoracic ganglia may be shown in
the early embryogenesis by the result of cell counting. However, it should be stressedout that the number of neuroblasts in the mandibular ganglia is smaller than that of
the maxillary and labial ganglia. This difference of the number of neuroblasts ingnathal ganglia may be connected with structural differences, that is, the mandibular
segment bears a pair of simple appendages, while the maxillary and labial segments
bear complicated appeendages with palps.
In the abdominal segments, no appendages are formed, except the pleuropodia in
the first abdominal segment, so that the significant structural differences within abdominal segments do not occur. However, the number of neuroblasts in the 10th abdominal segment is extremely small, and it is likely that this segment expresses its ownfunctional specialization or difference.
According to Miyamoto and Shimozawa (1983) and Miyamoto (1983), the segmental homology of the ganglia is expressed in the early neurogenesis, while the segmental specialization of ganglia is expressed in the late neurogenesis, because each segment becomes to express its own functional specialization or difference with the advance of development, in contrast to the structural homologies. In K. tibialis the segmental specialization of ganglia, of the mandibular and the 10th abdominal segments,
occurs in the early neurogenesis.

Acknowledgements
I wish to express my cordial thanks to Prof. H. Ando, Sugadaira Montane Research Center, University of Tsukuba, for his guidance as well as invaluable suggestionand encouragement given to me in the course of study.

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223

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Author's address: Dr. T. Kishimoto
Kansai Sogo Kankyo Center, Nakazaki-Nishi 2-3-39, Kita, Oosaka 530,
Japan