76 (1): 65 – 86
14.5.2018© Senckenberg Gesellschaft für Naturforschung, 2018.

Egg structure and embryonic development ofarctoperlarian stoneflies: a comparative embryo­logical study (Plecoptera)
Shodo Mtow & Ryuichiro Machida
Sugadaira Research Station, Mountain Science Center, University of Tsukuba, Sugadaira Kogen, Ueda, Nagano 386-2204, Japan; Shodo
Mtow [s610@sugadaira.tsukuba.ac.jp]; Ryuichiro Machida [machida@sugadaira.tsukuba.ac.jp]
Accepted 08.i.2018.
Published online at www.senckenberg.de/arthropod-systematics on 30.iv.2018.
Editors in charge: Benjamin Wipfler & Klaus-Dieter Klass
Abstract. Egg structure and embryonic development of nine arctoperlarian stoneflies from nine families, i.e., Scopuridae, Taeniopterygidae, Leuctridae, Capniidae, and Nemouridae of Euholognatha, and Perlidae, Chloroperlidae, Perlodidae, and Peltoperlidae of Systello­
gnatha were examined and compared with previous studies. The primary aim of this study was to use embryological data to reconstruct
the groundplan and phylogeny of Plecoptera and Polyneoptera. Euholognatha has eggs characterized by a thin, transparent chorion, while
the eggs of Systellognatha are characterized by a collar and anchor plate at the posterior pole. These features represent an apomorphic
groundplan for each group. The embryos form by the concentration of blastoderm cells toward the posterior pole of the egg. Soon after
the formation of the embryo, amnioserosal folds form and fuse with each other, resulting in a ball-shaped “embryo-amnion composite”
that is a potential autapomorphy of Plecoptera. As an embryological autapomorphy of Polyneoptera, embryo elongation occurs on the egg
surface, supporting the affiliation of Plecoptera to Polyneoptera. After its elongation on the egg surface, the embryo sinks into the yolk with
its cephalic and caudal ends remaining on the egg surface. This unique embryonic posture may be regarded as an apomorphic groundplanof Plecoptera. Arctoperlarian plecopterans perform three types of katatrepsis: 1) the first type, in which the embryo’s anteroposterior anddorsoventral axes change in reverse during katatrepsis, is found in Capniidae, Nemouridae, Perlidae, Chloroperlidae, and Perlodidae, andthis sharing is symplesiomorphic; 2) the second one, in which the embryo’s axes are not changed during katatrepsis, is found in Scopuridae, Taeniopterygidae, and Leuctridae, and this may be regarded as synapomorphic to them; 3) the third one, in which the embryo rotatesaround its anteroposterior axis by 90° during katatrepsis as known for Pteronarcyidae, is found in Peltoperlidae, and this type may be
synapomorphic to these two families.
Key words. Scopuridae, Euholognatha, Systellognatha, Arctoperlaria, Polyneoptera, comparative embryology, egg structure, blastokinesis.

1.

Introduction

Plecoptera or stoneflies are a hemimetabolous, neopteranorder, occurring on all continents excluding Antarctica,
and more than 3,700 species have been described, including fossil species (DeWalt et al. 2015). Nymphs arealmost exclusively aquatic and can be found mainly in
cold, well-oxygenated running waters. Stoneflies are important components of clean streams, and they are often
used as bioindicators (Fochetti & Tierno de Figueroa
2008).
Plecoptera are divided into the two suborders, Antarctoperlaria and Arctoperlaria. The former is found onlyin the Southern Hemisphere and contains four families.

ISSN 1863-7221 (print)| eISSN 1864-8312 (online)

The Arctoperlaria inhabit mostly the Northern Hemisphere, comprising two subgroups, Systellognatha andEuholognatha, each containing six families (Zwick 2000;
Beutel et al. 2014; DeWalt et al. 2017). Recent molecular phylogenetic analyses support the monophyly ofeach suborder and each arctoperlarian subgroup (Terry
2004; McCulloch et al. 2016). However, in contrast to
Antarctoperlaria, the monophyly of Arctoperlaria is only
supported by morphological characters related to thecomplex mate-finding syndrome “drumming,” which isshared by all families of this group (with the exception of
the Scopuridae) (Zwick 1973, 2000).

65Mtow & Machida: Comparative embryology of Arctoperlaria

Polyneoptera comprise ten lower neopteran orders,
including the Plecoptera, Dermaptera, Embioptera,
Phasmatodea, Orthoptera, Zoraptera, Grylloblattodea,
Mantophasmatodea, Mantodea, and Blattodea (= “Blattaria” + Isoptera). However, phylogenetic relationships
among these orders have been much disputed over (e.g.,
Kristensen 1975; Boudreaux 1979; Hennig 1981; Klass
2009; Beutel et al. 2013). In addition, the monophyly of
Polyneoptera has been debated over for a long time, but
it was recently supported based on morphological andembryological studies (e.g., Yoshizawa 2011; Mashimo
et al. 2014; Wipfler et al. 2015) and molecular data (e.g.,
Ishiwata et al. 2011; Song et al. 2016); Misof et al. (2014)
conducted a large-scale phylogenomic analysis based ontranscriptomes of 1,478 genes, and provided a strong support for monophyletic Polyneoptera. Phylogenetic positions of Zoraptera and Dermaptera, both of which has
long been under debate (see Klass 2003, 2009; Beutel& Weide 2005), were reliably placed in the monophyletic Polyneoptera (e.g., Ishiwata et al. 2011; Yoshizawa
2011; Mashimo et al. 2014; Misof et al. 2014). In spite ofrecent challenges from various disciplines, phylogenetic
relationships within Polyneoptera remain still far apartfrom consensus (see Beutel et al. 2013).
Plecoptera, of which affiliation to Polyneoptera seems
currently established, remain a challenging taxonomic
group in the Neoptera (Zwick 2009). Various hypotheses have been proposed for the phylogenetic position of
Plecoptera including sistergroup relations to the remaining neopterans (e.g., Kristensen 1975; Beutel & Gorb
2006; Klug & Klass 2007; Zwick 2009), to the remaining polyneopterans (Paurometabola or Pliconeo­pterain­clud­ing Zoraptera, cf. Wipfler et al. 2015) (Fausto etal. 2001; Beutel et al. 2014) and even to Paraneo­pteras.l. (= Acercaria + Zoraptera) + Holometabola (Ross
1955; Hamilton 1972). Recent comparative morphologies, molecular phylogenetics, and combined analyses
have proposed different polyneopteran orders or assemblages as the sister group of Plecoptera, including
the “Dermaptera” (Ishiwata et al. 2011), “Embioptera”(Kukalová-Peck 2008), “Orthoptera” (Kômoto et al.
2012), “Zoraptera” (Matsumura et al. 2015), “Zoraptera+ Dermaptera” (Terry & Whiting 2005), “Zoraptera +Embioptera” (Grimaldi & Engel 2005), “Chimaeraptera(= Xenonomia = Grylloblattodea + Mantophasmatodea)”(Blanke et al. 2012), “Orthoptera + Chimaeraptera + Eukinolabia (= Embioptera + Phasmatodea) + Dictyoptera(= Mantodea + Blattodea)” (Misof et al. 2014). However, these recent changes were not always based on newevidence for Plecoptera itself, but Plecoptera was only
shoved around as other taxa were studied and views of
their interrelations changed (Zwick 2009).
A comparative embryological approach can be a potential source of deep phylogenetic information that canhelp to resolve these debates. However, our knowledgeof the embryonic development of Plecoptera is limited.
Although several detailed studies exist in the arctoperlarian subgroup Systellognatha, these come from only twoof six families: Pteronarcys proteus Newman, 1838 of
66

Pteronarcyidae by Miller (1939, 1940) and Kamimuria
tibialis (Pictet, 1841) of Perlidae by Kishimoto & Ando
(1985, 1986) and Kishimoto (1986, 1987). Little data exist on the development of other systellognathan families
and Euholognatha (Khoo 1968a,b; Kishimoto 1997), andembryological information on Antarctoperlaria is totallylacking.
	On this background, we started a comparative embryological study of Plecoptera. In the present study, we
describe an outline of embryogenesis for all the nine Japanese arctoperlarian families (Kawai 1967; Shimizu et al.
2005): five families of the infraorder Euholognatha, i.e.,
Scopuridae, Taeniopterygidae, Leuctridae, Capniidae,
and Nemouridae; four families of the infraorder Systellognatha, i.e., Perlidae, Chloroperlidae, Perlodidae, andPeltoperlidae with special reference to external morpho­
logy, and compare the results with previous works, todiscuss the groundplan of Plecoptera and the interfamily
relationships in Arctoperlaria.

2.

Materials and methods

Adults from nine arctoperlarian stoneflies (Table 1) werecollected in 2014 to 2016 around the streams in Sugadaira Kogen, Ueda, Nagano, Japan, i.e., Daimyojin-zawa,Kara-sawa, and Naka-no-sawa. As for Scopura mon­tana, late instar larvae were reared and raised to adults
en masse in plastic cases (167 mm × 117 mm × 58 mm)
containing stones and a layer of water and fed with fallen
leaves.
Females after mating were kept separately in plastic
cases (68 mm × 39 mm × 15 mm) containing tissue paper
under controlled temperature (Table 1), and fed on fruits(apple or persimmon) and commercial food for insects(Mushi-jelly, Mitani, Ibaraki, Japan) and fish (TetraFin,
Spectrum Brands Japan, Yokohama, Japan, or Koi-no-sato, Japan Pet Food, Tokyo, Japan). The eggs deposited byfemales were incubated in plastic cases (36 mm × 36 mm
× 14 mm) filled with water under controlled temperature(Table 1).
Prior to fixation, eggs were soaked in commercial
bleach (Kitchen Bleach S, Mitsuei, Fukushima, Japan)
for several seconds and cleaned using a small brush toremove the gelatinous layer that covered them. The eggs
were rinsed in Ephrussi-Beadle’s solution (0.75% NaCl,
0.035% KCl, 0.021% CaCl2) containing detergent (0.1%
Triton X-100), punctured with a ﬁne needle, ﬁxed witheither Kahle’ s fixative (ethyl alcohol : formalin : acetic
acid : distilled water = 15 : 6 : 2 : 30) or FAA (ethyl alcohol : formalin : acetic acid = 15 : 5 : 1) for 24 h, andstored in 80% ethyl alcohol at room temperature.
The ﬁxed eggs were stained with DAPI (4’,6-diamidino-2-phenylindole dihydrochloride) solution diluted to
10 µg/l with PBS (18.6 mM NaH2PO4 : H2O, 84.1 mM
NaH2PO4 : 2H2O, 1.75 M NaCl; pH 7.4) at 4°C for
20 min to several days depending on specimens. The eggs

ARTHROPOD SYSTEMATICS & PHYLOGENY — 76 (1) 2018

Table 1. The nine Japanese arctoperlarian stoneflies of which egg structure and embryonic development were examined in the present
study.

Materials

Month of oviposition

Temperature for incubation of
adults and eggs

Scopuridae: Scopura montana Maruyama, 1987
Taeniopterygidae: Obipteryx Okamoto, 1922 sp.
Leuctridae: Paraleuctra cercia (Okamoto, 1922)
Capniidae: Apteroperla tikumana (Uéno, 1938)
Nemouridae: Protonemura towadensis (Kawai, 1954)
Systellognatha

October – December
June
May  –  June
February  –  April
November  –  December

8°C
12°C
12°C
4°C
8°C

Perlidae: Calineuria stigmatica (Klapálek, 1907)
Chloroperlidae: Sweltsa Ricker, 1943 sp.
Perlodidae: Ostrovus Ricker, 1952 sp.
Peltoperlidae: Yoraperla uenoi (Kohno, 1946)

September  –  October
May  –  June
June  –  July
June  –  July

12°C
12°C
12°C
12°C

Euholognatha

stained with DAPI were observed under a ﬂuorescencestereomicroscope (MZ FL III + FluoCombi, Leica, Heerbrugg, Switzerland) with UV excitation at 360 nm. Systellognatha eggs, which have a tough chorion layer, were
dechorionated with a fine needle and fine forceps prior tostaining.
For scanning electron microscopy (SEM) observation, eggs were postﬁxed with 1% OsO4 for 1 h. Fixed
eggs were dehydrated in a graded ethanol series, dried
either with a critical point dryer (Samdri-PVT-3D, tousimis, Rockville, Maryland) or naturally dried with HMDS
(1,1,1,3,3,3-Hexamethyldisilazane) as described by
Faull & Williams (2016), coated with gold, and thenobserved under an SEM (SM-300, TOPCON, Tokyo,
Japan) at 15 kV. Eggs of Apteroperla tikumana, which
are prone to distortion in the course of processing due
to their softness, were observed using the nano-suit
method, as described by Takaku et al. (2013) and Fujita
et al. (2016). Eggs were soaked in 1% polyoxyethylene
sorbitan monolaurate (Tween 20) solution for 1 h, blotted brieﬂy on dry ﬁlter paper to remove excess solution,
mounted on a stab, and observed with the SEM at 5 kV.
To record blastokinesis, some eggs were observed
while alive using a time-lapse VTR system (CK-2 orCK-40, inverted microscope, Olympus, Tokyo, Japan;
TSN401A, CCD color camera, Elmo, Nagoya, Japan;
Live capture 2, web camera system, downloaded fromhttp://www2.wisnet.ne.jp/~daddy).

3.

Results

The orientation of the insect eggs is defined according to
the embryo just before hatching (Wheeler 1893). When
applying this definition to plecopterans, we encounter aserious problem. As generally found in hemimetabolan
insects, i.e., Palaeoptera, Polyneoptera, and Acercaria,
usually in plecopterans, 1) the embryo forms at the posterior pole of the egg or the ventral side near the posterior

pole; 2) in the course of anatrepsis, the embryo substantially elongates with its posterior end ahead, resulting
in its ventral side facing the dorsal side of the egg andthe embryo’s anteroposterior axis reversed, i.e., both theanteroposterior and dorsoventral axes of the embryo become opposed to those of the egg; 3) katatrepsis then occurs, and the embryo appears again on the egg surface,shifting its position to the ventral side of the egg, and its
anteroposterior axis is reversed, i.e., both the anteroposterior and dorsoventral axes of the embryo correspondagain to those of the egg (see Anderson 1972; Mashimo
et al. 2014). This type of blastokinesis was revealed tooccur in a large proportion of the plecopterans examined in the present study such as Apteroperla tikumana(Capniidae) (Figs. 9, 16: see also 3.1.4.). However, anaberrant form of katatrepsis, in which the embryo maintained unchanged positions of the anteroposterior anddorsoventral axes, was found to be performed in a small
proportion of the plecopterans examined such as Scopura
montana (Scopuridae) (Figs. 5, 16: see also 3.1.1.). In
these plecopterans, the embryo reaches hatching, with its
anteroposterior and dorsoventral axes opposed to thoseof other plecopterans. Simply following to the generaldefinition of the orientation of eggs by Wheeler (1893),
we would have to describe, for example, that in these
plecopterans the embryo forms at the anterior pole of theegg, which would thus differ from other plecopterans. To
avoid such a problem in orientation, in the present study
we define the orientation of the egg in Plecoptera as follows: 1) the posterior is where the embryo forms, and theanterior is its opposite; 2) the dorsal is where the embryo
exists just before katatrepsis, and the ventral is the opposite.
In what follows, we describe the egg structure and an
outline of embryonic development in one species fromeach of nine arctoperlarian families. As for the embryonic development, first we made a detailed description
on S. montana, dividing it into 12 stages following Kishimoto & Ando (1985), and then we gave descriptions forother species, focusing on the differences from S. mon­tana and/or other species.
67Mtow & Machida: Comparative embryology of Arctoperlaria

Fig. 1. Eggs of Euholognatha. SEM micrographs. A – D. Scopura montana; A: egg, lateral view, anterior at the top, B: enlargement of theegg surface near the anterior pole; endochorion can be seen through a tear of the exochorion, C: egg surface of the anterior pole, D: enlargement of C, showing a rosette, at the center of which a micropyle is seen. E,F. Obipteryx sp., lateral view, anterior at the top; E: egg(some lines on egg surface are artifacts), F: enlargement of a micropylar area. G: egg of Paraleuctra cercia, which is artificially wrinkled
during the drying for processing specimens, lateral view. H: egg of Apteroperla tikumana, lateral view, anterior at the top. I,J. Protonemura
towadensis; I: egg, which is artificially dented during the drying for processing specimens, lateral view, anterior at the top, J: micropyles(white slanting line is an artifact). — Abbreviations: Ench, endochorion; Exch, exochorion; arrowhead, micropyle. Scale bars = A, 100 µm;
B,D,F,J, 10 µm; C,E,G – I, 50 µm.

68ARTHROPOD SYSTEMATICS & PHYLOGENY — 76 (1) 2018

Fig. 2. Eggs of Systellognatha. SEM micrographs. A – E. Calineuria stigmatica; A: egg, with the anchor plate resolved by soaking inbleach for a short time, lateral view, anterior at the top, B: enlargement of the posterior pole, C: posterior pole, with the anchor plate
resolved, D: anterior half of egg, showing micropyles, E: enlargement of micropyles. F – H. Sweltsa sp.; F: egg, lateral view, anterior at
the top, G: anterior half of the egg, H: micropyles. I – N. Ostrovus sp.; I: right side of the egg, anterior at the top, J: left side of the egg,
K: newly laid egg, ventral view, left side to the top, anterior to the right, L: egg just before hatching, ventral view, M: posterior half of theleft side of the egg, N: enlargement of micropyles. O – R. Yoraperla uenoi; O: egg, anterior view, P: egg, posterior view, Q: egg, lateral view,
R: enlargement of micropyles. — Abbreviations: AP, anchor plate; Co, collar; arrowhead, micropyle. Scale bars = A,D,F,I – L,O – Q,
100 µm; B,C,G,M, 50 µm; E,H,N,R, 10 µm.

69Mtow & Machida: Comparative embryology of Arctoperlaria

Fig. 3. Embryonic development of Scopura montana, Stage 1. DAPI staining, A – F. lateral view, anterior at the top, G – L. posterior view.
A,G: cleavage, late stage, B,H: blastoderm, C – E,I – K: differentiation of embryonic and extraembryonic areas, beginning (C,I), middle(D,J) and late (E,K) stages, F,L: newly formed germ disc. — Abbreviations: Bd, blastoderm; BdC, blastoderm cell; CN, cleavage nucleus;
EA, embryonic area; EeA, extraembryonic area; GD, germ disc; Se, serosa; SeC, serosal cell. Scale bars = 100 µm.

70ARTHROPOD SYSTEMATICS & PHYLOGENY — 76 (1) 2018

3.1.

Egg and embryonic development of
Euholognatha

3.1.1. Scopura montana (Scopuridae)
Egg. Eggs are spheroidal with long and short diametersof 330 – 400 µm and 300 – 330 µm, respectively (Fig.
1A). They are ivory in color because the yellowish yolk isvisible through the transparent egg membranes. The surface is surrounded by a sticky coat. The egg membranes
are composed of a two-layered chorion comprising an
exochorion and endochorion (Fig. 1B): the former often
peels off during embryonic development, and the latter
is furnished with numerous rod-like materials about 1 – 2µm in length on its surface (Fig. 1B). The exochorion of
the anterior third of the egg contains a weak, polygonal
pattern, which forms several rosettes around the anteriorpole of the egg (Fig. 1C): at the center of each rosette, amicropyle of ca. 2 µm in diameter opens, and the micropyles are arranged roughly in circle (Fig. 1C,D).
Embryonic development. The egg period is 75 – 85 days
at 8°C.
Stage 1: The cleavage nuclei arrive at the surface ofthe yolk mass (Fig. 3A,G), and a unicellular blastoderm
forms (Fig. 3B,H). Soon after completion of blastoderm
formation, the embryonic and extraembryonic areas aredifferentiated. The former forms at the posterior poleof the egg and is more densely cellulated than the latter(Fig. 3C,I), and the nuclei of the former are smaller andmore crowded than those of the latter (Fig. 3D,E,J,K).
The embryonic area is differentiated into a discoid germdisc about 100 µm in diameter, and the extraembryonicarea is the serosa (Figs. 3F,L, 5A, 6A).
Stage 2: The amnion is produced from the margin of
the germ disc or embryo (Fig. 4A). The amnion forms
the amnioserosal fold along with the serosa, and anatrepsis starts. The amnioserosal fold, of which formation ismore progressive in the posterior region of the embryothan in the anterior, extends beneath the embryo (Fig.
4B – D). Finally, the amnioserosal folds fuse with eachother, and the amniotic pore is closed. After the fusion of
the amnioserosal folds, the embryo is elliptical, with longand short diameters approximately 120 µm and 85 µm,
respectively (Fig. 4D), but then becomes circular about
110 µm in diameter (Figs. 5B, 6B).
Stage 3: The embryo elongates approximately to 185µm in length along the posterior egg surface (Figs. 5C,
6C). The protocephalon and protocorm differentiate, andthe embryo assumes a pear-shape (Fig. 6C). As a result of
fusion of amnioserosal folds, the egg surface is entirely
covered with the serosa, and the serosal cuticle starts tobe secreted beneath the chorion.
Stage 4: The embryo elongates posterior to ap­pro­xima­tely 350 µm along the egg surface, attaining morethan one-third of the egg circumference (Fig. 5D). The
protocephalon grows wider into the form of a head lobe
(Fig. 6D). Segmentation starts at this stage (not distinctly
shown in figures). The serosal cuticle forms a thickening
at the posterior pole of the egg (cf. SeCt in Fig. 6D).

Fig. 4. Embryonic development of Scopura montana, Stage 2.
DAPI staining, posterior view, anterior of embryo at the top. A –  D:
formation of amnioserosal fold, successive stages A to D. — Abbreviations: AmC, amniotic cell; AmP, amniotic pore; ASF, amnioserosal fold; Em, embryo; Se, serosa; SeC, serosal cell. Scale bars
= 100 µm.

Stage 5: The embryo elongates posteriorly with its
caudal region ahead, and its anteroposterior axis is reversed (Fig. 5E). The thoracic to anterior abdominal region sinks into the yolk with the cephalic and posterior
abdominal regions remaining on the egg surface (Figs.
5E, 6E). The embryo then assumes an S-shape. Segmentation proceeds towards the posterior, and appendages
develop in the differentiated segments. The stomodaeum
appears at the center of the head (Fig. 6E).
Stage 6: The embryo grows and further sinks into theyolk (Fig. 5F). Segmentation and appendage formation
continue to proceed. The clypeolabrum appears as a single swelling anterior to the stomodaeum. Antennae andgnathal appendages are clearly visible (Fig. 6F).
Stage 7: The embryo grows in the yolk, attaining its
maximum length (Figs. 5G, 6G).
Stage 8: The embryo develops further, with the abdomen enlarged in the yolk, and the head moving close to
the egg surface (Figs. 5H, 6H). The appendages develop,
and in the maxilla, the palp, coxopodite, galea, and lacinia can be distinguished (Figs. 5H, 6H).
Stage 9: In this stage, katatrepsis occurs. The am­nio­se­rosal fold ruptures around the region where the amnio­
tic pore closed, and the embryo appears again on the egg
surface (Figs. 5I, 6I). The serosa migrates toward theventral region of the egg and condenses to form the secondary dorsal organ. The amnion spreads over the area
the serosa had occupied, functioning as the provisional
dorsal closure (Figs. 5I, 6I). The embryo does not change
its position during katatrepsis, and its positioning during
intertrepsis on the dorsal side of the egg is maintained,71

Mtow & Machida: Comparative embryology of Arctoperlaria

Fig. 5. Embryonic development of Scopura montana. DAPI staining, lateral view, anterior at the top, ventral to the left. A: Stage 1, B: Stage2, C: Stage 3, D: Stage 4, E: Stage 5, F: Stage 6, G: Stage 7, H: Stage 8, I: Stage 9, J: Stage 10, K: Stage 11, L: Stage 12. — Abbreviations:Am, amnion; An, antenna; Ce, cercus; CE, compound eye; Cllr, clypeolabrum; Em, embryo; Ga, galea; GD, germ disc; HC, head capsule;
HL, head lobe; La, lacinia; Md, mandible; MxCp, maxillary coxopodite; MxP, maxillary palp; Pce, protocephalon; Pco, protocorm; SDO,
secondary dorsal organ; Se, serosa; Th1L, first thoracic leg. Scale bars = 100 µm.

with its head kept at the posterior pole of the egg. As a result, the anteroposterior and dorsoventral axes of the embryo remain opposite to those of the egg, and the embryoreaches hatching keeping this orientation (Figs. 5I – L,
15A – C). The cephalic and thoracic appendages and cerci
are well developed: in the labial appendages, the palp andthe glossa-paraglossa complex are distinguishable (Figs.
5I, 6I).
Stage 10: The embryo further grows and the definitive dorsal closure proceeds from the posterior (Figs. 5J,
6J). The secondary dorsal organ enlarges.
Stage 11: Definitive dorsal closure is almost complete, and the head capsule acquires its definitive form
(Figs. 5K, 6K). The clypeolabrum differentiates into theclypeus and labrum. The frons becomes distinct. A transparent embryonic cuticle is secreted, and the cuticle on
the frons forms a sclerotized egg tooth. The compoundeyes appear (Fig. 6K).
Stage 12: The larval cuticle is secreted, and theembryo acquires the configuration of the first instar
larva (Figs. 5L, 6L). The full-grown embryo tears thechorion and serosal cuticle using the egg tooth and hatches out.
72

3.1.2. Obipteryx sp. (Taeniopterygidae)
Egg. Eggs are spheroidal with long and short diameters
approximately 230 µm and 170 µm, respectively (Fig.
1E). The chorion is thin and transparent. A pair of micropylar areas is on both lateral sides at the level of equator,each of which has three to four micropyles ca. 2 µm in
diameter with a hood which is the chorionic extension
overhanging the micropyle (Fig. 1F).
Embryonic development. The egg period is approximately 125 days, including the diapause period of twomonths, at 12°C. The embryonic development of Obi­
pteryx sp. closely resembles that in Scopura montana ingeneral aspects, but in Obipteryx sp. the germ disc approximately 20 µm in diameter is much smaller than thatof S. montana, even when considering its smaller egg
size (Stage 1, Fig. 7A). Soon after the amnioserosal folds
fuse with each other at the onset of anatrepsis, development enters diapause for approximately 60 days (Stage2, Fig. 7B). When diapause terminates, the embryoelongates along the posterior egg surface (Stage 3, Fig.
7C). The embryo elongates posterior with its caudal end
ahead, attaining more than one-third of the egg circum-ARTHROPOD SYSTEMATICS & PHYLOGENY — 76 (1) 2018

Fig. 6. Embryonic development of Scopura montana. DAPI staining, posterior view, ventral at the top. A: Stage 1, B: Stage 2, C: Stage 3,
D: Stage 4, E: Stage 5, F: Stage 6, G: Stage 7, H: Stage 8, I: Stage 9, J: Stage 10, K: Stage 11, L: Stage 12. — Abbreviations: An, antenna;
CE, compound eye; Cl, clypeus; Cllr, clypeolabrum; Em, embryo; ET, egg teeth; Fr, frons; Ga, galea; GD, germ disc; GPC, glossa-paraglossa complex; HC, head capsule; HL, head lobe; La, lacinia; LbP, labial palp; Lr, labrum; Md, mandible; MxCp, maxillary coxopodite;
MxP, maxillary palp; Pce, protocephalon; Pco, protocorm; SDO, secondary dorsal organ; Se, serosa; SeCt, thickening of serosal cuticle;
Th1, first thoracic segment. Scale bars = 100 µm.

ference (Stage 4, Fig. 7D). The anterior abdomen (Stage5, Fig. 7E), followed by the thoracic region, sinks into
the yolk with the cephalic region and posterior abdomen
remaining on the egg surface, and the embryo assumes an
S-shape (Stages 6 – 8, Fig. 7F – H). The embryo orientation is opposite that of the egg. Katatrepsis (Stage 9, Fig.
7I) and development in post-katatrepsis stages (Stages
10 – 12, Fig. 7J – L) of Obipteryx sp. are similar to thosein S. montana, and the embryo reaches hatching with its
orientation contrary to that of the egg.

mately 40% of the egg circumference (Stages 3, 4, Fig.
8C,D). The following elongation of the embryo and its
positioning in the yolk are similar to those shown in thestoneflies described above (Stages 5 – 8, Fig. 8E – H). The
egg and embryo are orientated opposite of each other.
Katatrepsis (Stage 9, Fig. 8I) and development in postkatatrepsis stages (Stages 10 – 12, Fig. 8J – L) are similar
to those of the two species described above, and the embryo reaches hatching with its orientation contrary to thatof egg.

3.1.3. Paraleuctra cercia (Leuctridae)

3.1.4. Apteroperla tikumana (Capniidae)

Egg. Eggs are spheroidal with long and short diameters
approximately 140 µm and 120 µm, respectively (Fig.
1G). The chorion is thin and transparent. No data wereobtained on micropyles.
Embryonic development. The egg period is 45 – 55 days
at 12°C. The embryonic development of Pa. cercia basically resembles those of the stoneflies described above.
The embryo formed (Stages 1, 2, Fig. 8A,B) posteriorly
elongates with its caudal end ahead, attaining approxi-

Egg. Eggs are spheroidal with long and short diameters
approximately 170 µm and 135 µm, respectively (Fig.
1H). The chorion is thin and transparent. No data wereobtained on micropyles.
Embryonic development. The egg period is approximately 55 days at 4°C. Prior to katatrepsis, embryonicdevelopment of A. tikumana resembles the species shown
above (Stages 1 – 8, Fig. 9A – H), with few minor differences regarding the positioning of the embryo. The early73Mtow & Machida: Comparative embryology of Arctoperlaria

Fig. 7. Embryonic development of Obipteryx sp. DAPI staining, lateral view, anterior at the top, ventral to the left. A: Stage 1, B: Stage 2,
C: Stage 3, D: Stage 4, E: Stage 5, F: Stage 6, G: Stage 7, H: Stage 8, I: Stage 9, J: Stage 10, K: Stage 11, L: Stage 12. — Abbreviations:Am, amnion; An, antenna; Ce, cercus; CE, compound eye; Em, embryo; GD, germ disc; HC, head capsule; HL, head lobe; Md, mandible;
MxP, maxillary palp; Pce, protocephalon; Pco, protocorm; SDO, secondary dorsal organ; Se, serosa; Th1L, first thoracic leg. Scale bars =100 µm.

embryo grows also anteriorly (Stages 3, 4, Fig. 9C,D),
and the cephalic end of the embryo attains approximately
the middle of the ventral side of the egg (Stages 6 – 8,
Fig. 9F – H). The immersion of the embryo into the yolk
in Stage 5 is restricted to the caudal region of the abdomen (Fig. 9E). In katatrepsis, differently from the three
above-mentioned stoneflies, the embryo appeared on theegg surface moves along the egg surface with its headahead, passing the posterior pole of the egg, then on the
egg’s ventral side toward the anterior pole. Consequently,
the anteroposterior and dorsoventral axes of the embryo,which had been in an opposite orientation to those of
the egg during intertrepsis (e.g., Stage 8, Fig. 9H), arereversed and now correspond to those of the egg. The
serosa is condensed dorsoposterior to the head, and thesecondary dorsal organ is formed on the dorsal side of theegg (Stage 9, Fig. 9I). The embryo maintains its orientation in accord with the egg, and the embryo continues
developing and reaches hatching (Stages 10 – 12, Fig.
9J – L).

74

3.1.5. Protonemura towadensis (Nemouridae)
Egg. Eggs are spheroidal, with long and short diameters
approximately 150 µm and 130 µm, respectively (Fig.
1I). The chorion is thin and transparent. In the egg, twomicropyles approximately 2 µm in diameter are locatedon the equator (Fig. 1J).
Embryonic development. The egg period is approximately 45 days at 8°C. The embryonic development of
Pr. towadensis (Fig. 10A – L) resembles that of Apter­
operla tikumana in general aspects (Fig. 9A – L), withminor differences regarding the positioning of the embryo. In Pr. towadensis, the embryo extends less anteriorly (Fig. 10E – H), and the immersion of the embryo inStage 5 is more extensive relative to A. tikumana (Fig.
9E). Katatrepsis occurs in Stage 9 (Fig. 10I), and as in
A. tikumana, the anteroposterior and dorsoventral axesof the embryo become to correspond to those of the egg(Stages 9 – 12, Figs. 10I – L, 15D – F).

ARTHROPOD SYSTEMATICS & PHYLOGENY — 76 (1) 2018

Fig. 8. Embryonic development of Paraleuctra cercia. DAPI staining, lateral view, anterior at the top, ventral to the left. A: Stage 1, B:
Stage 2, C: Stage 3, D: Stage 4, E: Stage 5, F: Stage 6, G: Stage 7, H: Stage 8, I: Stage 9, J: Stage 10, K: Stage 11, L: Stage 12. — Abbreviations: Am, amnion; An, antenna; Ce, cercus; CE, compound eye; Cllr, clypeolabrum; Em, embryo; Ga, galea; GD, germ disc; HC,
head capsule; HL, head lobe; LbP, labial palp; Lr, labrum; Md, mandible; MxCp, maxillary coxopodite; MxP, maxillary palp; Pce, protocephalon; Pco, protocorm; SDO, secondary dorsal organ; Se, serosa; Th1L, first thoracic leg. Scale bars = 50 µm.

3.2.

Embryonic development of
Systellognatha

Due to the thick, tough chorion of Systellognatha eggs,
the chorion was removed prior to observing embryonicdevelopment. However, it is very difficult to remove thechorion from Stage 1 eggs because the serosal cuticle isyet to be secreted. Therefore, with the exception of Yora­perla uenoi (Peltoperlidae), which has an anteroposteriorly flattened egg, we could not observe Stage 1 in theremaining Systellognatha.
3.2.1. Calineuria stigmatica (Perlidae)
Egg. The eggs are spheroidal with long and short diameters approximately 550 µm and 400 µm, respectively(Fig. 2A). The chorion is smooth and fuscous in color. At
the posterior pole of the egg, the chorion is modified intoa collar-shaped protrusion (Fig. 2A,C), which is covered
with an adhesive attachment apparatus, known as the anchor plate (anchor, anchor base, or basal plate) (Fig. 2B).
At one third from the anterior pole of the egg, 10 – 15 mi-cropyles about 5 µm in diameter are arranged in a circle(Fig. 2A,D,E).
Embryonic development. The egg period is 200 – 250
days, including the diapause period of three months, at
12°C. Embryonic development basically resembles thoseof the stoneflies described above, especially those stoneflies whose embryos’ axes reversed during katatrepsis.
When the embryo is about 90 µm in diameter, it forms
at the ventral side near the posterior pole (Stage 2, Fig.
11A), and the embryos soon enter diapause for approximately 90 days. When diapause terminates, the embryo
starts to elongate (Stage 3, Fig. 11B) with the protocephalon and protocorm differentiated, and the embryo continues to elongate along the egg surface until it extends toapproximately 200 µm and covers more than one-third of
the egg circumference (Stage 4, Fig. 11C). The anterior
abdomen sinks into the yolk in Stage 4, and gnathal andthoracic regions follow in Stages 5 – 6 (Fig. 11D,E). The
embryo develops and acquires an S-shape, with the headand posterior abdomen remaining on the egg surface(Stages 7, 8, Fig. 11F,G). Katatrepsis occurs in Stage 9.
The embryo reverses its anteroposterior and dorsoventral

75Mtow & Machida: Comparative embryology of Arctoperlaria

Fig. 9. Embryonic development of Apteroperla tikumana. DAPI staining, lateral view, anterior at the top, ventral to the left. A: Stage 1, B:
Stage 2, C: Stage 3, D: Stage 4, E: Stage 5, F: Stage 6, G: Stage 7, H: Stage 8, I: Stage 9, J: Stage 10, K: Stage 11, L: Stage 12. — Abbreviations: Am, amnion; An, antenna; Em, embryo; GD, germ disc; HC, head capsule; HL, head lobe; Lb, labium; Md, mandible; Mx,maxilla; MxP, maxillary palp; Pce, protocephalon; Pco, protocorm; SDO, secondary dorsal organ; Se, serosa; Th1L, first thoracic leg. Scalebars = 50 µm.

axes, and the orientation of the embryo corresponds tothat of the egg (Fig. 11H). Keeping its orientation according with that of the egg, the embryo continues to develop
and then hatches (Stages 10 – 12, Fig. 11I – K).
3.2.2. Sweltsa sp. (Chloroperlidae)
Egg. The eggs are spheroidal with long and short diameters approximately 400 µm and 250 µm, respectively(Fig. 2F). The chorion is smooth and light yellow incolor. The specialized structures, including the collar andanchor plate, are lacking. On a third of the anterior partof the egg, six to nine micropyles about 5 µm in diameterare arranged in a circle (Fig. 2G,H).
Embryonic development. The egg period is 50 – 65 days
at 12°C. The embryonic development resembles those of
the stoneflies described above, especially those stoneflies
whose embryos’ axes are reversed in katatrepsis. An embryo of approximately 100 µm in diameter forms at theposterior pole of the egg, which looks thick because of alarge curvature around the posterior egg pole (Stage 2,
Fig. 12A). The embryo elongates along the egg surface
in Stages 3 to 4 (Fig. 12B,C), and the anterior abdomen76

sinks into the yolk with the cephalic and thoracic regions
and posterior abdomen remaining on the egg surface
in Stage 5 (Fig. 12D). Development continues throughStages 6 – 8, with the embryo retaining this posture (Fig.
12E – G). As a result of katatrepsis occurred in Stage 9,
the embryo reverses its anteroposterior and dorsoventral
axes, and the orientation of the embryo corresponds tothat of the egg (Fig. 12H). Maintaining this orientation,
the embryo continues to develop and reaches hatching(Stages 10 – 12, Fig. 12I – K).
3.2.3. Ostrovus sp. (Perlodidae)
Egg. The eggs are light yellow in color and unique inshape (Fig. 2I – L). Newly laid eggs are limpet-like inshape and are flattened laterally, i.e., from side to side,
their left side being less convex (Fig. 2I – K). As development proceeds, the left side of the egg swells (Fig. 2L).
The posterior refers to the direction where the collar andanchor plate exist, and the anterior is opposite: the anchor
plate is on the posterior end of the left side (Fig. 2I) andthe collar is on the right side (Fig. 2J). When observing
the egg from its right side as shown in Fig. 2I, the ventral

ARTHROPOD SYSTEMATICS & PHYLOGENY — 76 (1) 2018

Fig. 10. Embryonic development of Protonemura towadensis. DAPI staining, lateral view, anterior at the top, ventral to the left. A: Stage1, B: Stage 2, C: Stage 3, D: Stage 4, E: Stage 5, F: Stage 6, G: Stage 7, H: Stage 8, I: Stage 9, J: Stage 10, K: Stage 11, L: Stage 12. —Abbreviations: Am, amnion; An, antenna; Ce, cercus; CE, compound eye; Em, embryo; GD, germ disc; HC, head capsule; HL, head lobe;
Md, mandible; MxCp, maxillary coxopodite; MxP, maxillary palp; Pce, protocephalon; Pco, protocorm; SDO, secondary dorsal organ; Se,
serosa; Th1L, first thoracic leg. Scale bars = 50 µm.

side is to the right hand and the dorsal side to the left.
The length (anteroposterior length) of the egg is ca. 400µm, the width (dorsoventral length) is ca. 320 µm (Fig.
2I,J), and the thickness is ca. 150 µm just after oviposition (Fig. 2K), and ca. 220 µm just before hatching (Fig.
2L). The chorion shows a weak polygonal pattern on its
left side and toward the posterior (Fig. 2M,N). Along theequator of the left side of the egg, five to eight longitudinal micropyles are arranged, each with a width of 3 µm
(Fig. 2J,M,N).
Embryonic development. The egg period is approximately 80 days at 12°C. In the representations of embryogenesis in the above descriptions, the ventral side is to theleft (Figs. 5, 7 – 12). However, it is difficult to place theeggs with their convex dorsal side down in this species.
Therefore, embryogenesis is presented in Fig. 13 with theventral side of the egg to the right. The embryo ca. 50 µm
in diameter forms around the posterior pole (Stage 2, Fig.
13A). However, due to the unique shape of the egg, thenewly formed embryo is positioned a little biased to theright side of the egg, as shown in Fig. 13A. The embryoelongates partially twisted (Fig. 13B – F), and the embryos, which have not largely grown within the egg, are seen

as if they are sunk in the yolk (Fig. 13B – G). Therefore,
while it is difficult to precisely compare embryonic development of Ostrovus sp. with the other species, it is clearthat it resembles other plecopterans, especially those
whose embryonic axes are reversed during katatrepsis.
Namely, the formed embryo elongates along the dorsal
surface of the egg with its caudal end ahead, and the embryo’s anteroposterior and dorsoventral axes become opposed to those of the egg (Fig. 13B – G). Katatrepsis thenoccurs in Stage 9 (Fig. 13H). The embryo reverses its anteroposterior and dorsoventral axes, and the orientation of
the embryo eventually corresponds to that of the egg (Fig.
13H). The embryo grows further and hatches out from theegg, tearing the egg membrane around the anterior thirdof the right side of the egg (Fig. 13I – K).
3.2.4. Yoraperla uenoi (Peltoperlidae)
Egg. The eggs are reddish-brown. Initially the eggs arestrongly flattened anteroposteriorly, with a diameter andthickness of approximately 400 µm and 150 µm, respectively (Fig. 2O – R), but as development progresses, they
expand to about 240 µm. A transparent anchor plate is on77

Mtow & Machida: Comparative embryology of Arctoperlaria

Fig. 11. Embryonic development of Calineuria stigmatica. DAPI staining, lateral view, anterior at the top, ventral to the left. A: Stage 2,
B: Stage 3, C: Stage 4, D: Stage 5, E: Stage 6, F: Stage 7, G: Stage 8, H: Stage 9, I: Stage 10, J: Stage 11, K: Stage 12. — Abbreviations:Am, amnion; An, antenna; Ce, cercus; CE, compound eye; Em, embryo; Ga, galea; HC, head capsule; HL, head lobe; LbP, labial palp; Lr,labrum; Md, mandible; MxCp, maxillary coxopodite; MxP, maxillary palp; Pce, protocephalon; Pco, protocorm; SDO, secondary dorsalorgan; Se, serosa; Th1L, first thoracic leg. Scale bars = 100 µm.

the posterior side of egg, but the collar is inconspicuous(Fig. 2P,Q). The anterior side of the egg has a honeycomb pattern (Fig. 2O – Q). On the anterior side of theegg five to eight micropylar protuberances of several microns in diameter are roughly arranged in a circle, and a
micropyle approximately 1.5 µm in diameter opens at thecenter of each protuberance (Fig. 2O,R).
Embryonic development. The egg period is approximately 40 days at 12°C. Due to difficultly observing theextremely flattened eggs from the lateral side, we represent embryogenesis in Y. uenoi using photos from theposterior side (Fig. 14), which differs from the other species (Figs. 5, 7 – 13). The germ disc forms at the posteriorpole (Stage 1, Fig. 14A). In Stage 2, anatrepsis begins
and the amnioserosal folds fuse with each other (Fig.
14B). The embryo begins to elongate in an invertedtriangular shape, with the protocephalon and protocorm
differentiated (Stage 3, Fig. 14C), and then forms into aslug-like shape (Stage 4, Fig. 14D). Segmentation andappendage formation commence, and the anterior abdomen sinks into the yolk with the cephalic and thoracic
regions and posterior abdomen remaining on the egg
surface (Stage 5, Fig. 14E). The embryo then acquires an
78

S-shape (in sections, but not shown here) (Stages 6 – 8,
Fig. 14F – H). Katatrepsis occurs, the embryo slips outof the yolk (Stage 9, Fig. 14I), and is put down sideways
with its right side down, changing its posture from warped
to ventrally bent (Stage 10, Fig. 14J). Keeping this condition, the embryo further develops (Stage 11, Fig. 14K)
and hatches from the egg, tearing the egg membrane atits lateral side (Stage 12, Fig. 14L).

4.

Discussion

4.1.

Egg

Zwick (1973, 2000) suggested that: 1) the sclerotized
hard chorion is a groundplan character of Plecoptera,being universally present in Antarctoperlaria and systellognathan Arctoperlaria; 2) the soft chorion is likely
apomorphic to euholognathan Arctoperlaria; whereas 3)
systellognathan Arctoperlaria retain the hard chorion,
which differentiates into a collar surrounding the adhe-ARTHROPOD SYSTEMATICS & PHYLOGENY — 76 (1) 2018

Fig. 12. Embryonic development of Sweltsa sp. DAPI staining, lateral view, anterior at the top, ventral to the left. A: Stage 2, B: Stage 3, C:
Stage 4, D: Stage 5, E: Stage 6, F: Stage 7, G: Stage 8, H: Stage 9, I: Stage 10, J: Stage 11, K: Stage 12. — Abbreviations: Am, amnion;
An, antenna; Ce, cercus; CE, compound eye; Em, embryo; ET, egg teeth; Ga, galea; GPC, glossa-paraglossa complex; HC, head capsule;
HL, head lobe; Lb, labium; LbP, labial palp; Lr, labrum; Md, mandible; Mx, maxilla; MxCp, maxillary coxopodite; MxP, maxillary palp;
Pce, protocephalon; Pco, protocorm; SDO, secondary dorsal organ; Se, serosa; Th1,3L, first and third thoracic legs. Scale bars = 100 µm.

sive attachment apparatus (anchor plate) at the posteriorpole of the egg. The collar and anchor plate represent theapomorphic groundplan of Systellognatha, because as
Hinton (1981) pointed out, these features are not foundin other plecopterans, i.e., Antarctoperlaria and Euholo­gnatha, nor in other Neoptera.
Examining the egg structures of nine Japanese arc­to­perlarians, i.e., five species for Euholognatha – Sco­pu­ramontana (Scopuridae), Obipteryx sp. (Taenioptery­gi­dae),Paraleuctra cercia (Leuctridae), Apteroperla tikumana(Capniidae) and Protonemura towadensis (Nemouridae) – and four species for Systellognatha – Calineuria
stigmatica (Perlidae), Sweltsa sp. (Chloroperlidae), Os­
trovus sp. (Perlodidae) and Yoraperla uenoi (Peltoperlidae) – the present study corroborates Zwick’s (1973,
2000) understanding of arctoperlarian eggs. We characterize the eggs of Euholognatha and Systellognatha as
follows, referring to previous studies as necessary. The
eggs of Euholognatha are: 1) spherical or ellipsoid inshape (Fig. 1A,E,G,H,I); 2) without specialized structures such as a collar or anchor plate (Fig. 1A,E,G,H,I);and 3) covered by a thin, transparent chorion, which issmooth and without a conspicuous superficial pattern(Fig. 1E – J), although the exochorion of the anterior thirdof the egg wears a weak, polygonal network in S. mon­tana (Fig. 1B – D). The scopurid egg structure has been
described previously with “Scopura longa Uéno, 1929”
by Kawai & Isobe (1984), but it is likely that the materials examined at the time were in fact S. montana, as the
scopurids from this sampling site in Mt. Hachibuse, Nagano Prefecture, were more recently identified as S. mon­tana (see Uchida & Maruyama 1987).
The eggs of the other arctoperlarian infraorder Systellognatha may be characterized as: 1) spherical or ellipsoidal (Fig. 2A,F), but they sometimes take a specificshape characteristic of each group (Fig. 2I – N,O – Q); 2)
equipped with a collar and anchor plate on their posterior pole (Fig. 2A – C,I – N,O – Q); and 3) covered by athick and hard, colored chorion occasionally containing conspicuous superficial patterns or sculptures (Fig.
2I – N,O – Q) (Knight et al. 1965a,b). The eggs of thechloroperlid species, Sweltsa sp., lack the collar and anchor plate. However, because these structures are foundpredominantly in Systellognatha, and chloroperlid genera are known to include species with and without thesestructures (Stark et al. 2015), the absence of these struc79

Mtow & Machida: Comparative embryology of Arctoperlaria

Fig. 13. Embryonic development of Ostrovus sp. DAPI staining, lateral view from right side, anterior at the top, ventral to the right. A:
Stage 2, B: Stage 3, C: Stage 4, D: Stage 5, E: Stage 6, F: Stage 7, G: Stage 8, H: Stage 9, I: Stage 10, J: Stage 11, K: Stage 12. — Abbreviations: Am, amnion; An, antenna; Ce, cercus; CE, compound eye; Em, embryo; ET, egg teeth; Ga, galea; HC, head capsule; HL, headlobe; La, lacinia; Lr, labrum; Md, mandible; MxCp, maxillary coxopodite; MxP, maxillary palp; Pce, protocephalon; Pco, protocorm; SDO,
secondary dorsal organ; Se, serosa; Th1L, first thoracic leg. Scale bars = 100 µm.

tures in Sweltsa sp. may be due to a secondary modification (Fig. 2F).
In the present study, we observed the micropyles ofthree euholognathan stoneflies: S. montana, Obipteryxsp., and Pr. towadensis (Fig. 1C,D,F,J). In S. montana,several micropyles are distributed in a circle in a rosettepattern around the anterior pole of the egg (Fig. 1C,D). In
Obipteryx sp., micropylar areas with three to four micropyles are located on the equator on both lateral sides ofthe egg (Fig. 1F), as reported for another taeniopterygid
Brachyptera trifasciata (Pictet, 1832) (Degrange 1957),
and this micropylar arrangement may be characteristic of
the Taeniopterygidae. Two micropyles are located on theequator in Pr. towadensis (Fig. 1J), while several micropyles were distributed along the equator and in the posterior half of the egg in another Protonemura, Pr. praecox(Morton, 1894) (Degrange 1957). Although we failed
to detect micropyles in A. tikumana, Kishimoto (1997;
personal comm.) reported two micropyles located on thelateral side of the egg.
With the exception of Ostrovus sp., the micropyles of
Systellognatha were arranged in a circle (Fig. 2A,D,G,O).
A similar pattern of micropyle distribution has been
80

found in Pteronarcys proteus, in the Pteronarcyidae(Miller 1939), as well as in other systellognathan re­presentatives (Stark & Stewart 1981; Isobe 1988), withthe circular arrangement being a part of the groundplan
of Systellognatha. In Ostrovus sp., eggs have a laterallyflattened shape and a unique arrangement of micropyles,
with several micropyles arranged in a straight line on theleft side of the egg (Fig. 2J,M,N). This unusual micropylar arrangement in this species may be due to a secondary
modification related to its unique egg shape.
Additional studies in other species, especially the
Antarctoperlaria, are required to reconstruct the groundplan of micropylar distributions in the Plecoptera andArctoperlaria. However, the circular arrangement of
micropyles is quite likely a part of the groundplan of
Systellognatha. Moreover, given that one of the euholognathan families Scopuridae also shows a circular arrangement of micropyles, this feature may be regarded
as a potential groundplan of Arctoperlaria. Potential
explanations for the absence of a circular arrangementof micropyles in the remaining Euholognatha species
include a partial interruption of the micropylar arrangement or a reduction of the micropyles, i.e., the micro-

ARTHROPOD SYSTEMATICS & PHYLOGENY — 76 (1) 2018

Fig. 14. Embryonic development of Yoraperla uenoi. DAPI staining, posterior view. A: Stage 1, B: Stage 2, C: Stage 3, D: Stage 4, E: Stage5, F: Stage 6, G: Stage 7, H: Stage 8, I: Stage 9, J: Stage 10, K: Stage 11, L: Stage 12. — Abbreviations: Ab, abdomen; Am, amnion; An,antenna; Ce, cercus; CE, compound eye; Cllr, clypeolabrum; Em, embryo; Ga, galea; GD, germ disc; GPC, glossa-palaglossa complex;
HC, head capsule; HL, head lobe; La, lacinia; Lb, labium; LbP, labial palp; Md, mandible; Mx, maxilla; MxP, maxillary palp; Pce, protocephalon; Pco, protocorm; SDO, secondary dorsal organ; Se, serosa; Th1L, first thoracic leg. Scale bars = 100 µm.

pyles may have been lost in Obipteryx sp. on the dorsal and ventral sides of the egg, and most of those may
have been reduced in Pr. towadensis and A. tikumana.
However, explaining the extraordinary arrangement of
micropyles reported for Pr. praecox will require a reexamination of micropylar arrangement in this and otherrelated species.

4.2.

Embryonic development

4.2.1. Formation of the embryo
Mashimo et al. (2014) compared embryogenesis in He­
mi­metabola, and proposed two embryological autapomorphies of Polyneoptera. One involves elongation of
the embryo, as we discuss below in the section “4.2.2.1.
Anatrepsis and elongation of the embryo,” and the otheris on the manner of the embryo’s formation. In Polyneo­
ptera, the embryo is formed by the fusion of paired blastoderm regions with higher cellular density: Dermaptera(Shimizu 2013), Embioptera (Jintsu 2010), Phasmatodea (Bedford 1970), Orthoptera (Miyawaki et al. 2004),
Zoraptera (Mashimo et al. 2014), Grylloblattodea (Uchi-

fune & Machida 2005), and Blattodea (Fujita & Machida2017). However, in the Palaeoptera and Acercaria (e.g.,
Ephemeroptera: Tojo & Machida 1997; Odonata: Ando
1962; Psocodea: Goss 1952; Thysanoptera: Heming1979), blastoderm cells around the posterior pole concentrate in one area and proliferate to form the embryo.
This type of germ disc formation is also known for theapterygote Ectognatha, i.e., Archaeognatha (Machida etal. 1990) and Zygentoma (Masumoto & Machida 2006),
clearly suggesting that this is a plesiomorphic condition
to Pterygota. Consequently, the formation of the embryo
or germ disc by the fusion of paired blastoderm areas
with higher cellular density, may be regarded as an apomorphic groundplan of Polyneoptera.
Information on the formation of embryo in Plecoptera
is fragmentary. Only two embryological analyses exist
for Systellognatha, including Miller (1939) for Pter­
onarcys proteus (Pteronarcyidae) and Kishimoto (1986)
for Kamimuria tibialis (Perlidae). In P. proteus, a small
germ disc is formed by the direct migration of a cellgroup appeared in the yolk on to the blastoderm. In K.
tibialis, a small germ disc is formed by the simple migration of blastoderm cells. These imply that the embryos
form in Plecoptera without involving the fusion of paired81Mtow & Machida: Comparative embryology of Arctoperlaria

blastoderm areas with higher cellular density, which isdifferent from other groups of Polyneoptera. Thus, in thepresent study, we examined embryo formation in fiveeuholognathan and four systellognathan arctoperlarians,
employing DAPI staining, with special reference to theeuholognathan S. montana. We demonstrated that theembryo is formed by the concentration and proliferationof blastoderm cells around the posterior pole (Fig. 3), as
Kishimoto (1986) observed for the systellognathan, K.
tibialis. Specifically, in Plecoptera the embryo is formed
not in the manner involving the fusion of paired blastoderm areas with higher cellular density, which Mashimo
et al. (2014) proposed as an apomorphic groundplan feature of Polyneoptera, but in a simple concentration andproliferation of blastoderm cells, as shown in apterygote
Ectognatha, Palaeoptera and Acercaria, that may be taken to be plesiomorphic to Pterygota. As described in “1.
Introduction,” the phylogenetic position of Plecoptera
has been debated, but recent comparative morphologies
and phylogenomics (e.g., Beutel et al. 2014; Misof et al.
2014) have often bestowed basal positions to Plecoptera
within Polyneoptera. The manner of embryo formationin Plecoptera, which seems unique in Polyneoptera, isexpected to be critically discussed with respect to phylogenetic reconstruction of Polyneoptera.
In addition, embryo formation in P. proteus occurs
as a compact cellular aggregation beginning early in development. However, according to Miller (1939), thestreaming of a cellular group from inside to the peripheryof the egg is involved in embryo formation. Such a centripetal migration of a mass of presumptive embryoniccells is a singular example reported in insects, and criticalreexamination is needed.
4.2.2. Blastokinesis
According to Fujita & Machida (2017), we define terms
related to blastokinesis as follows. Embryos of Insectas.str. (Ectognatha: Archaeognatha, Zygentoma, andPterygota), immerse in the yolk in the early stage of development due to the formation of amnioserosal folds.
The embryos then elongate and take their final position in
the pre-katatrepsis period. The entire descending processof the embryo from commencement of the amnioserosal
fold formation up to this point, is the “anatrepsis.” After
anatrepsis, the embryos develop until katatrepsis occurs,
maintaining this positioning, this phase being the “intertrepsis.” The rupture and withdrawal of the amnioserosal
folds then occur, which leads to the embryo’s reappearance on the egg surface, this ascending process being the“katatrepsis.” These processes related to developmental
phase are collectively the “blastokinesis.” In the present
study, we examined blastokinesis in five euholognathan
and four systellognathan arctoperlarians, focusing on theeuholognathan Scopura montana.
4.2.2.1. Anatrepsis and elongation of the embryo. As
soon as the germ disc or embryo forms at the posteriorpole of the egg, the marginal region begins to extend over82

the embryo, forming the amnioserosal folds, and anatre­
psis starts (Fig. 4A – C). The amnioserosal folds soon fusewith each other (Fig. 4D), and the amniotic pore is completely closed. Thus, in the earliest stage of development,
fusion of amnioserosal folds occurs and a compact, ballshaped “embryo-amnion composite” forms, of which thedorsal and ventral constituents are represented by theembryo proper and amnion, respectively. This process isthe same as that reported previously in Pteronarcys pro­teus (Miller 1939) and Kamimuria tibialis (Kishimoto &Ando 1985; Kishimoto 1986). The fusion of amnioserosal folds in the earliest stage of development, which leads
to the formation of a ball-shaped embryo-amnion composite, is unique to Plecoptera within the Polyneo­ptera
and could be a potential autapomorphy of this group.
The formed embryos elongate along the dorsal side ofthe egg with their posterior end ahead. After this elongation on the egg surface, the middle part of the embryos
curve and sink into the yolk, with their cephalic and caudal ends remaining on the egg periphery (Figs. 5C – E,
6C – E, 7C – E, 8C – E, 9C – E, 10C – E, 11B – D, 12B – D,
13B – D, 14C – E), as described in previous embryological studies on Plecoptera (Miller 1939, 1940; Kishimoto& Ando 1985; Kishimoto 1997). Thus, in Plecoptera, asin the other polyneopteran orders, including Dermaptera(Heymons 1895; Shimizu 2013), Embioptera (e.g., Kershaw 1914), Phasmatodea (e.g., Bedford 1970), Orthoptera (e.g., Roonwal 1937), Zoraptera (Mashimo et
al. 2014), Grylloblattodea (Uchifune & Machida 2005),
Mantophasmatodea (Machida et al. 2004), Mantodea(Hagan 1917), Blattodea (Heymons 1895; Fujita &Machida 2017), and Isoptera (e.g., Knower 1900), theformation of amnioserosal folds ends at an earlier stageof development and the elongation of the embryo occurring on the egg surface. Mashimo et al. (2014) suggested
this feature as another embryological autapomorphy of
Polyneoptera, taking it into considerations that in Palaeoptera and Acercaria, the embryo elongates, keeping stepwith its immersion into the yolk and with the formationof the amnioserosal folds: i.e., Ephemeroptera (Tojo &Machida 1997), Odonata (Ando 1962), Psocoptera (Goss
1952), Phthiraptera (Schölzel 1937), Thysano­ptera(Heming 1979), and Hemiptera (Cobben 1968; Heming& Huebner 1994). The present study demonstrates thatembryos of Plecoptera elongate in a manner regarded
as autapomorphic to Polyneoptera, and the placementof Plecoptera among the Polyneoptera is strongly corroborated. In contrast, there is little support for the phylogenetic hypotheses that places Plecoptera outside of
Polyneoptera, i.e., those proposing the sister group relationship of Plecoptera with Neoptera or with “Paraneo­
ptera + Holometabola.”
4.2.2.2. Intertrepsis. In most Plecoptera, as a result ofanatrepsis (e.g., Fig. 5C – G), the anteroposterior and dorsoventral axes of the embryo become opposed to those of
the egg. The exceptions to this pattern are in Yoraperlauenoi (Fig. 14) and Pteronarcys proteus (Miller 1939),
in which free movement of the embryos during blastoki-

ARTHROPOD SYSTEMATICS & PHYLOGENY — 76 (1) 2018

Fig. 15. Time lapse images of katatrepsis in two plecopteran species, lateral view, anterior at the top, ventral to the left. A – C. Scopura
montana; A: just before katatrepsis, B: in katatrepsis, C: just after katatrepsis. D – F. Protonemura towadensis; D: just before katatrepsis,
E: in katatrepsis, F: just after katatrepsis. — Abbreviations: Ab, abdomen; HL, head lobe; SDO, secondary dorsal organ; Y, yolk. Scalebars A – C = 100 µm; D – F = 50 µm.

nesis may be limited due to their flattened egg shape (cf.
“4.2.2.3. Katatrepsis”).
As described above, the plecopteran embryos descend
into the yolk with their cephalic and caudal ends remaining on the egg periphery, and they keep this postureduring intertrepsis (Figs. 5E – G, 7E – G, 8E – G, 9E – G,
10E – G, 11D – F, 12D – F, 13D – F). Such a posture ofembryos in intertrepsis may be unique to the Plecoptera
within Polyneoptera (see the literature cited in the previous section 4.2.2.1) and may be regarded as a part of thegroundplan of Plecoptera.
4.2.2.3. Katatrepsis. In the present study, we examinedkatatrepsis of nine families of the arctoperlarian Pleco­
ptera, and distinguished three katatrepsis types.
Type 1  –  Katatrepsis begins, and the embryo appearson the egg surface. The embryo moves along the egg
surface with its head ahead, via the posterior pole of theegg. It then moves to the ventral side of the egg, toward
the anterior pole of the egg, and katatrepsis completes.
The anteroposterior and dorsoventral axes are reversedto those in intertrepsis. Among the plecopterans examined, the euholognathan Capniidae and Nemouridae, andthe systellognathan Perlidae, Chloroperlidae, and Perlodidae fall into this category (Figs. 9I, 10I, 11H, 12H,
13H, 15D – F). Another representative of the Perlidae,
Kamimuria tibialis also shows Type 1 katatrepsis (Kishimoto & Ando 1985).
Type 2  –  Different from Type 1 katatrepsis, the embryo does not change its orientation throughout the courseof katatrepsis, and its anteroposterior and dorsoventral

axes remain opposed to those of the egg. Among the pleco­
pterans examined, three euholognathan families Scopuridae, Taeniopterygidae, and Leuctridae, are categorized
in this type (Figs. 5I, 6I, 7I, 8I, 15A – C). In the study
on diapause in the taeniopterygid euholognathan Bra­chyptera risi (Morton, 1896), Khoo (1968b) provided
figures showing that this species performs Type 2 kata­
trepsis.
Type 3  –  In the peltoperlid systellognathan Yoraper­la uenoi, of which eggs are strongly flattened anteroposteriorly (Fig. 2O – Q), the embryo forms at the center ofthe broad bottom of the egg (Fig. 14A,B), and it grows
and elongates there (Fig. 14C – H). Katatrepsis occurs
subsequently (Fig. 14I), and the embryo rotates around
its anteroposterior axis by 90 degrees, lying sideways on
the bottom side of the egg (Fig. 14J). Katatrepsis of thistype is also found in the pteronarcyid systellognathan
Pteronarcys proteus, which has eggs with anterodorsally
flattened shape, as in Y. uenoi (Miller 1939, 1940).
Katatrepsis involving a reversion of the embryo’s
axes like Type 1 is predominant in non-holometabolan
Pterygota: i.e., in Palaeoptera: Ephemeroptera (Tojo &Machida 1997), and Odonata (Ando 1962); Polyneo­
ptera: Dermaptera (Heymons 1895; Shimizu 2013), Embioptera (e.g., Kershaw 1914), Phasmatodea (e.g., Bedford 1970), Orthoptera (e.g., Roonwal 1937), Zoraptera(Mashimo et al. 2014), Grylloblattodea (Uchifune &Machida 2005), Mantophasmatodea (Machida et al.
2004), Blattodea (Heymons 1895; Fujita & Machida2017), and Isoptera (e.g., Knower 1900); Acercaria: Psocoptera (Goss 1952), Phthiraptera (Schölzel 1937), Thy83

Mtow & Machida: Comparative embryology of Arctoperlaria

Fig. 16. Phylogenetic reconstruction of 10 arctoperlarian families of which katatrepsis types havebeen clarified, incorporating the current phylogenetic understanding (e.g., Zwick 2000), i.e., eachof Antarctoperlaria, Euholognatha, and Systellognatha is monophyletic (see text).

sanoptera (Heming 1979), and Hemiptera (Cobben 1968).
On the other hand, the apterygote Ectognatha, such as
the Archaeognatha (Machida et al. 1994) and Zygentoma (Masumoto & Machida 2006), do not follow one ofthese established types of katatrepsis involving the reversion of the embryo’s axes. Therefore, we conclude that
this type of katatrepsis is an apomorphic groundplan of
Pterygota, and the sharing of Type 1 katatrepsis by some
pleco­pteran lineages, such as the euholognathan Capniidae and Nemouridae, and the systellognathan Perlidae,
Chloroperlidae, and Perlodidae, can be referred to as
symplesiomorphic.
In contrast, the Type 2 and Type 3 forms of katatrepsis
are apparently derived features in Plecoptera. Interfamily
relationships in Euholognatha are not well understood(Zwick 2000; Terry 2004; Kjer et al. 2006), but the affinity of the euholognathan Scopuridae, Taeniopterygidae,
and Leuctridae is suggested, taking Type 2 katatrepsis fora synapomorphy of them. In Systellognatha, the monophyly of Peltoperlidae, Pteronarcyidae and Styloperlidae
is well supported (e.g., Zwick 2000). The sharing of bothType 3 katatrepsis and a flattened egg shape phenotype
by the former two families may reflect their phylogenetic
affinity.
Figure 16 depicts the distribution of the different katatrepsis types on the phylogeny of the 10 plecopteranfamilies for which katatrepsis type is known. Additional information on blastokinesis from more lineages of
Plecoptera, especially the Antarctoperlaria, is strongly
desired.

5.

Acknowledgments

We thank Dr. Shigekazu Tomizuka, Dr. Yuta Mashimo, Dr. Mari
Fujita, and Mr. Kazuki Kojima of Sugadaira Research Station,

84

Mountain Science Center, University of Tsukuba (SRS) for their
help in collecting material and rearing. We also deeply thank Dr.
Toru Kishimoto of the Tsukuba International University for his giving valuable information, and Drs. Benjamin Wipfler, Peter Zwick,
and anonymous reviewers for their helpful comments. This study
was financially supported by the Sasakawa Scientific Research
Grant from The Japan Science Society to SM and Grants-in-Aid
from the JSPS (Scientific Research C: 25440201 and Scientific Research B: 16H04825) to RM. This is a contribution by SRS.

6.

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