11Proc. Arthropod. Embryol. Soc. Jpn. 52, 11–14 (2019)Ⓒ 2019 Arthropodan Embryological Society of Japan
ISSN 1341–1527[SHORT COMMUNICATION]

Note on the Thickened Serosa and Serosal Cuticle Formed beneath theEmbryo in Scopura montana Maruyama, 1987 (Insecta, Plecoptera,Scopuridae)
Shodo MTOW and Ryuichiro MACHIDA
Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305–8572, Japan
Current address: Sugadaira Research Station, Mountain Science Center, University of Tsukuba, Ueda, Nagano 386–2204, Japan
E-mail: s610@sugadaira.tsukuba.ac.jp (SM)Miller (1939, 1940) described the thickened serosa that
forms beneath the embryo in the stonefly Pteronarcys proteus
Newman, 1838 (Pteronarcyidae), which secretes a bulky
cuticular layer. He named the thickened serosa and bulky serosal
cuticle the “grumulus” and “grumorium,” respectively. Similar
serosal structures were reported in plecopteran Kamimuria
tibialis (Pictet, 1841) (Perlidae) by Kishimoto and Ando (1985),
who named the thickened serosa “columnar serosal cells.”
However, reference to the serosal structures is fragmented,and our knowledge of them is insufficient. For example, there
is no information on the thickening of serosa. In our study,
we provide a preliminary description of the formation of thethickened serosa and serosal cuticle in the scopurid stonefly,
Scopura montana Maruyama, 1987 (Scopuridae).
We collected late-instar larvae of Scopura montanafrom the streams in Sugadaira Kogen, Ueda, Nagano, Japanand raised them to the adult stage in the laboratory. The eggs
deposited by females were ﬁxed with Karnovskyʼs ﬁxative(2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M HClsodium cacodylate buffer, pH 7.2) for 24 h and postﬁxed with 1%
OsO4 for 1 h. Fixed eggs were dehydrated in a graded ethanol
series and embedded in an epoxy resin (Low Viscosity Resin,Agar, Essex, UK). Serial semi-thin sectioning at a thicknessof 0.75 µm was performed using an ultra-thin microtome(MT-XL, RMC, Arizona, USA) equipped with a diamond knife(Histo Jumbo Diamond Knife, Diatome, Nidau, Switzerland),according to the methods described by Blumer et al. (2002).
Sections were stained using 0.1% toluidine blue solution andexamined under a biological microscope (Optiphoto-2, Nikon,Tokyo, Japan).
We followed the embryonic development of Scopura
montana until the completion of the thickened serosa andserosal cuticle. Soon after a small germ disc of ca. 100 µm
in diameter appeared at the posterior pole of the egg, theamnioserosal folds start to form (Mtow and Machida, 2018).
During the segregation of the amnion from the embryonicmargin, the serosa extends beneath the embryo, causing theformation of amnioserosal folds (Fig. 1). The formation of
the amnioserosal folds is more progressive in the posterior
region of the embryo than in the anterior region (Fig. 1). The
amnioserosal folds draw closer to each other as the amnion
progressively segregates and the serosa extends. Figure 2Aand B show the embryo with its amnioserosal folds just before
their fusion. The inner and outer elements of amnioserosal
folds or the amnion and serosa, both of which are massive, aredistinguished by a faint boundary between them (arrowheadsin Fig. 2A, B). Finally, the amnioserosal folds completely fusewith each other (Fig. 3), and the serosa entirely covers theegg surface. Almost simultaneously, the amnion and serosaof the amnioserosal folds detach from each other. As a result,both amnion and serosa become independent, thick cellular.
The embryo and amnion which ventrally covers the embryo or
the embryo-amnion composite assumes a compressed, ballshaped configuration (Fig. 3). Then the serosa, which had beena part of the amnioserosal fold, converges beneath the embryoamnion composite to form the “thickened serosa,” of whichcells acquire a radial arrangement in section (Fig. 4). Soon after
the fusion of the amnioserosal folds, the serosa has started tosecrete the serosal cuticle over the entire surface of the egg(Fig. 4). The serosal cuticle is densely secreted beneath theembryo-amnion composite by the thickened serosa, because
the apical surfaces of the thickened serosa have converged
there (Fig. 4). As the embryonic development progresses,
the embryo gradually elongates and the amnion attenuates(Fig. 5). The serosal cuticle becomes further bloated beneaththe thickened serosa to form the “thickened serosal cuticle”(Fig. 5), wherein the three zones that are distinguished: theinnermost transparent zone, an intermediate toluidine-philic
indented zone, and an outermost yellowish zone.
Our present study revealed that in Scopura montana,
the thickened serosa that forms beneath the embryo at theposterior pole of the egg is derived from the serosal elementof the amnioserosal fold and the thickened serosa heavily
deposits a dense, cuticular substance as the thickened serosal12S. MTOW AND R. MACHIDA

Figs. 1, 2 Sections of a Scopura montana embryo undergoing the formation of amnioserosal folds. The exochorion has been removed.
Fig. 1 A sagittal section of an embryo in the early phase of amnioserosal fold formation. Anterior to the left.
Fig. 2 Vertical sections of an embryo with amnioserosal folds just before fusion. A and B show serial sections at a distance of approximately
3 µm. Arrowheads show the boundaries between the amnion and serosa.
Am: amnion, ASF: amnioserosal fold, Em: embryo, Ench: endochorion, Se: serosa, Y: yolk. Scales = 20 µm.

THICKENED SEROSA BENEATH THE EMBRYO IN PLECOPTERA

13Figs. 3–5 Sections of Scopura montana embryos undergoing the formation of a thickening serosa and serosal cuticle. The exochorion hasbeen removed.
Fig. 3 A vertical section of an embryo with the amnioserosal folds that have just fused.
Fig. 4 A vertical section of an embryo with a completely formed thickened serosa.
Fig. 5 A sagittal section of an embryo with a completely secreted thickened serosal cuticle. Anterior to the left.
Am: amnion, Em: embryo, Ench: endochorion, Me: mesoderm, Se: serosa, SeCt: serosal cuticle, TSeCt: thickened serosal cuticle,
Y: yolk, YC: yolk cell. Scales = 20 µm.

14S. MTOW AND R. MACHIDAcuticle. The thickened serosa and thickened serosal cuticle inS. montana are apparently comparable to the grumulus andgrumorium in Pteronarcys proteus, respectively (Miller, 1939,1940), and the thickened serosa in S. montana is comparable tothe columnar serosal cells in Kamimuria tibialis (Kishimotoand Ando, 1985). Although surveys covering more plecopteranrepresentatives are needed, the thickened serosa and a
thickened serosal cuticle may be groundplan features ofPlecoptera.
Structures similar to the thickened serosa and/or
thickened serosal cuticle have been reported in several
polyneopteran lineages, for example, Orthoptera (Slifer,1938; Slifer and Sekhon, 1963), Grylloblattodea (Uchifune andMachida, 2005), Embioptera (Jintsu and Machida, 2009), andPhasmatodea (Jintsu et al., 2010). Miller (1940) associated thegrumulus and grumorium of P. proteus with the “hydropylarcells” and “hydropyle(s)” of a grasshopper Melanoplus
differentialis, respectively (Acrididae), which may enable
water uptake by the egg (Slifer, 1938; Slifer and Sekhon, 1963).
However, our understanding of these structures in Plecoptera
is insufficient to phylogenetically or physiologically comparethem with such structures in other polyneopterans. Therefore,
more information is needed concerning the development ofthese structures in Plecoptera.

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Acknowledgments: The present study was supported
by a Grant-in-Aid from the JSPS, Scientific Research B(JP16H04825) to RM; Research Fellow (JP18J10360) to SM.
This is a contribution from the Sugadaira Research Station,
Mountain Science Center, University of Tsukuba.

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