9Proc. Arthropod. Embryol. Soc. Jpn. 53, 9–13 (2021)Ⓒ 2021 Arthropodan Embryological Society of Japan
ISSN 1341–1527[ORIGINAL PAPER]

Thickened Serosa and Serosal Cuticle Formed beneath the Embryo in
Eight Arctoperlarian Stoneflies (Insecta, Plecoptera)
Shodo MTOW1, 2) and Ryuichiro MACHIDA1)
Sugadaira Research Station, Mountain Science Center, University of Tsukuba, Sugadaira Kogen 1278–294, Ueda, Nagano 386–
2204, Japan
2)
Current affiliation: Faculty of Symbiotic Systems Science and Technology, Fukushima University, Kanayagawa 1, Fukushima,
Fukushima 960–1296, Japan
E-mail: impulse610@gmail.com (SM)1)

Abstract
We examined the embryological development of arctoperlarian stoneflies belonging to the eight families, i.e., Taeniopterygidae,
Leuctridae, Capniidae, and Nemouridae of Euholognatha as well as Perlidae, Chloroperlidae, Perlodidae, and Peltoperlidae ofSystellognatha, all of which developed a thickened serosa and serosal cuticle beneath the embryo. By combining these results withthose of existing studies, it was revealed that the major lineages of Arctoperlaria share these structures and that they can be regarded
as embryological groundplan features of Arctoperlaria.

Introduction
Plecoptera represent a polyneopteran order, comprising
two suborders, Arctoperlaria, primarily from the Northern
Hemisphere, and Antarctoperlaria, exclusively from theSouthern Hemisphere. The monophyly of each suborderand two arctoperlarian subgroups, Euholognatha andSystellognatha, is supported by both morphological (Zwick,2000) and molecular phylogenetic evidence (McCulloch etal., 2016). The phylogenetic position of Plecoptera has been
debated for a long time (e.g., Kristensen, 1975; Zwick, 2009;
Beutel et al., 2014), but recent morphological (Yoshizawa,
2011; Wipfler et al., 2015) and molecular evidence, including
a large-scale phylogenomic analysis based on transcriptomesof protein-coding genes (Ishiwata et al., 2011; Misof et al.,2014; Song et al., 2016; Wipfler et al., 2019) revealed that
Plecoptera is a member of Polyneoptera. Our comparativeembryological study of Plecoptera (Mtow and Machida, 2018a)
also corroborated the placement of Plecoptera in Polyneoptera
based on the finding that the plecopteran embryos elongate
along the egg surface, and this feature represents one of thepolyneopteran embryological autapomorphies (Mashimo etal., 2014).
We have been conducting comparative embryologicalstudies of Plecoptera, aiming at the reconstruction of thegroundplan of Plecoptera and subsequently of Polyneoptera(Mtow and Machida, 2018a, b, 2019). The embryos of somestoneflies show thickening of the serosa beneath theembryo. Miller (1939, 1940) described the thickened serosa

and thickened cuticular layer secreted beneath it in thesystellognathan stonefly Pteronarcys proteus of Pteronarcyidae.
These thickened serosa and serosal cuticle being named
the “grumulus” and “grumorium,” respectively. Similarly,
serosal structures were reported in another systellognathan,
Kamimuria tibialis of Perlidae, by Kishimoto and Ando (1985),
who named the thickened serosa “columnar serosal cells.” In
the case of Euholognatha, we recently found that the thickened
serosa and serosal cuticle also develop beneath the embryo in
Scopura montana of Scopuridae and gave a detailed descriptionof their structure and development (Mtow and Machida, 2018b,
2019). In the current study, we examined the embryologicaldevelopment of the arctoperlarian stoneflies belonging to
the seven major families of which embryogenesis has nothitherto been examined, specifically, Taeniopterygidae,
Leuctridae, Capniidae, and Nemouridae of Euholognatha
as well as Chloroperlidae, Perlodidae, and Peltoperlidae ofSystellognatha, also including one representative species ofPerlidae, to discuss whether the thickened serosa and serosal
cuticle formed beneath the embryo are groundplan features ofArctoperlaria.
Materials and Methods
Adults of eight arctoperlarian stoneflies from the eightfamilies, i.e., Euholognatha: Obipteryx sp. of Taeniopterygidae,
Paraleuctra cercia of Leuctridae, Apteroperla tikumana ofCapniidae, Protonemura towadensis of Nemouridae andSystellognatha: Calineuria stigmatica of Perlidae, Sweltsa sp.

10

S. MTOW AND R. MACHIDA

of Chloroperlidae, Ostrovus sp. of Perlodidae, and Yoraperlauenoi of Peltoperlidae, were collected between 2014 and 2017from the streams in Sugadaira Kogen, Ueda, Nagano, Japan.
The deposited eggs were either ﬁxed with Kahleʼs fixative(ethyl alcohol : formalin : acetic acid : distilled water = 15 : 6: 2 :30) for 24 h and stored in 80% ethyl alcohol at room temperatureor fixed with Karnovskyʼs ﬁxative (2% paraformaldehyde and2.5% glutaraldehyde in 0.1 M HCl-sodium cacodylate buffer,
pH 7.2) for 24 h and stored in the buffer at 4 C.
The eggs ﬁxed with Kahleʼs fixative were dehydrated ina graded ethanol series, immersed in acetone, and embedded
in a methacrylate resin (Technovit 7100, Kulzer, Wehrheim,
Germany) in accordance with the protocol described by
Machida et al. (1994a, b). Semi-thin sections at a thicknessof 2 µm were cut using a semi-thin microtome (H-1500, BioRad, Hercules, USA), equipped with a tungsten carbide knife(Superhard Knife, Meiwafosis, Tokyo, Japan). Sections were
stained with 0.5% Delaﬁeldʼs hematoxylin for 12 h, 0.5% eosin
G for 1 h, and 0.5% fast green FCF 100% ethanol solution for 1
min.
The eggs ﬁxed with Karnovskyʼs ﬁxative were dehydrated
in a graded ethanol series, immersed in methyl oxirane, andembedded in an epoxy resin (Agar Low Viscosity Resin Kit,
Agar Scientific, Essex, UK). Serial, semi-thin sectioning at a
thickness of 0.75 µm was performed using an ultramicrotome(MT-XL, RMC, Arizona, USA) equipped with a diamond knife(Histo Jumbo, DiATOME, Nidau, Switzerland) according to
the methods described by Blumer et al. (2002). The sections
were then stained using 0.1% toluidine blue O solution for 1 h.
The stained sections were observed under a biologicalmicroscope (Optiphoto-2, Nikon, Tokyo, Japan) and captured
with a CCD camera (DS-Fi2, Nikon, Tokyo, Japan).
Results and Discussion
Staging used in the description is based on Mtow andMachidaʼs staging (2018a) for the embryogenesis of the
scopurid stonefly Scopura montana (Table 1).
In the case of the Euholognatha, in Obipteryx sp. of
Taeniopterygidae, in Stage 2, the small thickened serosa forms
beneath the embryo as in S. montana (Mtow and Machida,2018b, 2019) and Pteronarcys proteus (Millar, 1939, 1940) (Fig.

1A). Almost simultaneously, the serosa starts to secrete theserosal cuticle over the entire surface of the egg, and with theprogression of embryonic development, the serosal cuticle isthickened (Fig. 1B). The serosal cuticle is a slightly thicker
in the region beneath the thickened serosa (Fig. 1A, B). In
Paraleuctra cercia of Leuctridae, the thickened serosa is also
formed beneath the embryo at the stage of amnioserosal foldformation (Stage 2) and shows a radial cellular arrangement
in sections, as in S. montana and Pt. proteus (Fig. 2A). Soon,the serosal cuticle starts to be secreted, and with progressive
embryogenesis, it is gradually thickened up to the middle
stage of intertrepsis (around Stage 6). The serosal cuticle isextremely thickened beneath the thickened serosa (Fig. 2B).
The thickened serosa of Apteroperla tikumana of Capniidae
and Protonemura towadensis of Nemouridae forms beneath theembryo in the same manner as in Obipteryx sp. and Pa. cercia(Figs. 3A, 4A), with the serosal cuticle beneath the thickened
serosa bloated as the thickened serosal cuticle (Figs. 3B, 4B).
In Systellognatha, the thickened serosa and serosal
cuticle are formed beneath the embryo, as in Euholognatha.
Figures 5–8 present these structures of the definitive form in
Calineuria stigmatica of Perlidae, Sweltsa sp. of Chloroperlidae,
Ostrovus sp. of Perlodidae, and Yoraperla uenoi of Peltoperlidae,
respectively.
The current study revealed that all the eight arctoperlarianfamilies examined developed thickened serosa and thickened
serosal cuticle beneath the embryo. Hence, it was ascertainedthat the major lineages of Arctoperlaria, i.e., Euholognatha:
Taeniopterygidae, Leuctridae, Capniidae, Nemouridae,
and Scopuridae (Mtow and Machida, 2018b, 2019) and
Systellognatha: Perlidae (Kishimoto and Anto, 1985; herein),
Chloroperlidae, Perlodidae, Peltoperlidae, and Pteronarcyidae(Miller, 1939, 1940) possess thickened serosa and serosal
cuticle. The lineages still lacking information are only two
families, specifically Notonemouridae, which is a close relativeof Nemouridae, and Styloperlidae, which is regarded as thesister group of Peltoperlidae by Zwick (2000). Therefore, it
can be concluded that the thickened serosa and thickened
serosal cuticle should be counted as embryological groundplan
features of Arctoperlaria. Embryological information on
Antarctoperlaria, which is totally lacking, is needed.

Table 1 Developmental stages used in the present study based on Mtow and Machidaʼs staging (2018a).
StagesStage 1
Stage 2
Stage 3
Stage 4
Stage 5
Stage 6
Stage 7
Stage 8
Stage 9
Stage 10
Stage 11
Stage 12Developmental events
Cleavage; formation of blastoderm; differentiation of embryo
Formation of amnioserosal foldDifferentiation of protocephalon and protocorm
Elongation of embryo along the egg surface; commencement of segmentation
Immersion of embryo into the yolk; formation of appendages
Embryo assuming an S shape in the yolk
Embryo attaining its maximum length in the yolk
Head of embryo moving close to the egg surface
Regression of amnioserosal fold (katatrepsis); embryo appearing on the egg surface; formation of secondary dorsal organ
Progressive definitive dorsal closure
Completion of definitive dorsal closure; secretion of embryonic cuticle; formation of compound eye and egg tooth
Acquisition of definitive form by embryo; secretion of larval cuticle

SEROSAL STRUCTURES BENEATH ARCTOPERLARIAN EMBRYOS

11

Figs. 1–4 Thickened serosa and serosal cuticle beneath the embryo of four euholognathan stoneflies, chorion removed, anterior of the egg to the
top.
Fig. 1 Sections of embryos of Obipteryx sp. (Taeniopterygidae), methacrylate resin. A. A vertical section of an embryo in Stage 2. B. A sagittal sectionof an embryo in Stage 4, anterior of the embryo to the left.
Fig. 2 Sections of embryos of Paraleuctra cercia (Leuctridae), epoxy resin. A. A vertical section of an embryo in Stage 3. B. A sagittal section of a
thickened serosa beneath the embryo in Stage 6, anterior of the embryo to the left.
Fig. 3 Sagittal sections of embryos of Apteroperla tikumana (Capniidae), methacrylate resin, anterior of the embryo to the left. A. A section of anembryo in Stage 3. B. A section of an embryo in Stage 6.
Fig. 4 Sagittal sections of embryos of Protonemura towadensis (Nemouridae), epoxy resin. Eggs, which were fixed with Karnovskyʼs fixative, were
postfixed with 1% OsO4 for 1 h. A. A section of an embryo in Stage 3. B. A section of an embryo in Stage 7.
Am: amnion, AmC: amniotic cavity, Em: embryo, Me: mesoderm, Se: serosa, SeCt: serosal cuticle, TSe: thickened serosa, TSeCt: thickened
serosal cuticle, Y: yolk. Scales = 20 µm.

12Figs. 5–8
Fig. 5
Fig. 6
Fig. 7
Fig. 8

S. MTOW AND R. MACHIDA

Thickened serosa and serosal cuticle beneath the embryo of four systellognathan stoneflies, anterior of the egg to the top.
A vertical section of an embryo of Calineuria stigmatica (Perlidae) in Stage 2, methacrylate resin.
A sagittal section of an embryo of Sweltsa sp. (Chloroperlidae) in Stage 4, methacrylate resin, anterior of the embryo to the left.
A vertical section of an embryo of Ostrovus sp. (Perlodidae) in Stage 4, methacrylate resin.
A sagittal section of an embryo of Yoraperla uenoi (Peltoperlidae) in Stage 8, epoxy resin, anterior of the embryo to the left.
Am: amnion, AmC: amniotic cavity, Ch: chorion, Em: embryo, Se: serosa, SeCt: serosal cuticle, TSe: thickened serosa, TSeCt: thickened
serosal cuticle, Y: yolk. Scales = 20 µm.

Machida, R., T. Nagashima and H. Ando (1994a) Embryonic

Acknowledgments: The present study was supported by
a Grant-in-Aid from the JSPS, Research Fellow (JP18J10360)
to SM; Scientific Research B (JP16H04825); and Scientific
Research C (JP19K06821) to RM. This is a contribution fromthe Sugadaira Research Station, Mountain Science Center,
University of Tsukuba.

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segregation of a jumping bristletail, Pedetontus unimaculatus
Machida (Hexapoda, Microcoryphia), with a note on an automatic

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