Proc. Arthropod. Embryol. Soc. Jpn. 47, 5–10 (2012) OVARIAN STRUCTURES IN POLYDESMID DIPLOPODSⒸ 2012 Arthropodan Embryological Society of Japan
ISSN 1341–1527

5[ORIGINAL PAPER]

Comparative Study of Ovarian Structures in Polydesmid Diplopods(Diplopoda: Polydesmida) with Special Reference to the Arrangementof Germ Cells
Kensuke YAHATA
Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305–8572, Japan
E-mail: yahata@biol.tsukuba.ac.jp

Abstract
The ovarian structures of four polydesmid diplopods (Nedyopus tambanus, Oxidus gracilis, Epanerchodus orientalis, andEucondylodesmus elegans) are described with particular attention paid to the arrangement of germ cells within the ovary. A
pair of longitudinally oriented germ zones, containing oogonia and connecting growing oocytes via follicle epithelia, were
present within the dorso-lateral region of these polydesmid ovaries. Within each germ zone, oogonia and growing oocytes
were arranged according to their volume, with oogonia and younger oocytes located at the medial ends, and larger oocytes
located more laterally. In some species, ovarian epithelia of paired germ zones formed paired folds that extended toward theovarian lumen and made paired longitudinal grooves on the dorsal surface of the ovary along both sides of its median axis.
Orderly arrangement of germ cells is regarded as a distinct common ovarian feature among polydesmid diplopods. The germzone folds likely enable many oocytes to develop at once.

Introduction
The class Diplopoda is divided into two subclasses,
the Penicillata and the Chilognatha. The former is regarded
as the more primitive subclass with a single order, while
the latter is more advanced and is composed of 13 or 14
orders (Enghoff, 1984).
Previous studies have suggested that the ovarian
structure of chilognathan diplopods can be characterized
by the following features: 1) a single long, sac-like ovary; 2)a lateral pair of longitudinal germ zones for oogonial
proliferation and oocyte growth within the ovarianepithelium; and 3) follicular connections between thegrowing oocytes in the ovarian lumen and the germ zones(on the Glomerida: Yahata and Makioka, 1997; on the
Spirostreptida: Sareen, 1967; on the Julida: Kubrakiewicz,
1987, 1991a, 1991b; on the Polydesmida: Seifert, 1932;
Kubrakiewicz, 1987). Despite the presence of these
distinguishing characteristics, controversy remains
regarding the basic ovarian structure in polydesmid
diplopods. Previous studies have been consentaneous in
their assertion that polydesmid diplopods display “paired(tubular) ovaries” accompanied by growing oocytes on
their outer surfaces and surrounded by a “single common
ovitube” (Versluys and Demoll, 1923; Radhaklishnan andAdiyodi, 1977; Nair, 1981). Such composition is peculiar
among chilognathan diplopods.
In addition to the characteristics of the polydesmid

ovary mentioned above, Kubrakiewicz (1987) observed
the presence of an orderly arrangement of oocytesoriented according to oogenetic growth in the ventrolaterally paired germ zones of a polydesmid, Oxidus
gracilis (formerly Orthomorpha gracilis). A similar orderly
arrangement of germ cells according to volume wasobserved in the ovary of a glomerid diplopod, Hyleoglomeris
japonica, (Yahata and Makioka, 1997); however, according
to cladistic analysis by Enghoff (1984), the Glomerida are
one of the most primitive chilognathan orders and are only
distantly related to the more advanced Polydesmida.
Despite previous research by Kubrakiewicz (1987),
detailed information regarding the location andarrangement of germ cells in polydesmid ovaries is stilllacking.
The present study describes the ovarian structure infour polydesmid species. The order Polydesmida iscomposed of four suborders: the Chelodesmidea,
the Paradoxosomatidea, the Polydesmidea and theDalodesmidea (Hoffman, 1979). In previous studies, threeparadoxosomatidean species (Seifert, 1932; Radhaklishnan
and Adiyodi, 1977; Nair, 1981; Kubrakiewicz, 1987) and apolydesmidean species (Versluys and Demoll, 1923) havebeen examined. The present study examines twoparadoxosomatidean species, including those examined by
Kubrakiewicz (1987), Nedyopus tambanus and Oxidus
gracilis, and two polydesmidean species from different

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K. YAHATA

families, Epanerchodus orientalis (Polydesmidae) andEucondylodesmus elegans (Doratodesmidae). Special
attention is paid to the arrangement of germ cells within
ovaries in order to reveal any features common amongpolydesmids.
Materials and Methods
Adult of Nedyopus tambanus (Attems) were collectedfrom a vegetable garden in Nanto City, Toyama Prefecture.
Adults and subadults of Oxidus gracilis (Koch) werecollected from leaf litter at a site located at Mt. Tsukuba,
Ibaraki Prefecture. Adults of Epanerchodus orientalis(Attems) were collected from leaf litter gathered in Nanto
City, Toyama Prefecture, and Ueda City, Nagano
Prefecture. Adults of Eucondylodesmus elegans Miyosi
were collected from leaf litter gathered in Iwaki City,
Fukushima Prefecture.
Adult females of the four species were fixed in Bouinʼssolution for a few days after excision of the head parts witha razor blade in a physiological saline solution. Fixed
specimens were decalcified in a 5% formic acid, 70%
ethanol solution for a few days, dehydrated in a gradedethanol-n-butanol series, and cleared with 100% methyl
benzoate. After being embedded in paraffin, serial sectionsof 5 µm were stained using either Mayerʼs hematoxylin
and eosin, Heidenhainʼs azan, or McManusʼs alcian blue–
periodic acid Schiff (PAS)–hematoxylin technique.
Results
Nedyopus tambanus and Oxidus gracilis
Nedyopus tambanus and Oxidus gracilis, bothbelonging to the family Paradoxosomatidae, displayed very
similar structures in the reproductive systems of adultfemales.
The adult ovary in both species was comprised of asingle median, long, sac-like organ running between thealimentary canal and the ventral nerve cord. The ovary of
N. tambanus was present within the fifth through the 19th
body segments, while that of O. gracilis was present within
the fourth through the 18th segments. The ovarian wall ofboth species, which surrounded a wide ovarian lumen,consisted of an ovarian epithelium ranging from 1–4 µm inthickness (Figs. 1–4). A pair of germ zones was found in
the dorso-lateral region of the ovary in both species (Figs.
1, 3). The ovarian epithelium of each germ zone formed ashallow and narrow fold that extended toward the ovarian
lumen (Figs. 1–4), creating a longitudinal groove on thesurface of the ovary (arrows in Figs. 1–4).
Oogonia and several growing oocytes were arranged
in order of increasing volume starting at a medial positionand continuing into the lateral region of each folded germzone (Figs. 1–4). Within each folded germ zone, oogonia
and younger oocytes were located along the medial end,
nearest to the dorsomedian line of the ovary; larger
oocytes were located along the fold of the germ zone, first

towards the innermost region of the ovary, and thenmoving more laterally; and the largest oocytes werelocated nearest to the lateral end of each germ zone (Figs.
2, 4).
Oogonia approximately 8 µm in diameter, together
with several very early previtellogenic oocytes less than
10 µm in diameter, were observed among germ zone
epithelial cells (Figs. 2, 4). Growing oocytes larger than 10µm in diameter were surrounded by a layer of extremely
flattened follicle cells. The follicular epithelium of theseoocytes was connected with the ovarian epithelium of thegerm zones. Early previtellogenic oocytes larger than 20µm in diameter had consistently strong basophilic ooplasm
and one or two distinct nucleoli in their large germinal
vesicles (Figs. 2, 4). Larger oocytes in late previtellogenic
and vitellogenic stages were located more laterally, farfrom the oogonial area, in each germ zone.
Epanerchodus orientalis
The adult ovary in Epanerchodus orientalis wascomprised of a single median, long, wide and sac-like organ
extending from about the fourth to the 19th body segment.
The ovarian wall, by which a wide ovarian lumen was
surrounded, consisted of an ovarian epithelium ranging
from 1–7 µm in thickness (Figs. 5, 6). Ovarian epithelial
cells were somewhat flattened, each with a spheroidal
nucleus approximately 6 µm in diameter (Fig. 7).
A pair of germ zones was found in the dorso-lateralregion of the ovary (Fig. 5). The ovarian epithelium of eachgerm zone formed a deep and wide fold that extended
toward the ovarian lumen, creating a longitudinal grooveon the surface of the ovary (arrows in Figs. 5, 6).
Within each germ zone, oogonia and several growing
oocytes were arranged in order of increasing volume.
Oogonia as well as the smallest oocytes were located along
the medial end of the germ zone, nearest to thedorsomedian line of the ovary; larger oocytes wereoriented along the germ zone, first towards the innermost
region of the ovary, then moving laterally, towards thelateral end of the germ zone; and the largest oocytes werelocated at the most dorso-lateral position, opposite side to
the oogonial area (Figs. 5, 6). Oogonia approximately 10µm in diameter contained a nucleus with basophilic
chromatin granules but lacked a nucleolus (Fig. 7). Theseoogonia were only found in the region of the germ zone
nearest to the dorsomedian line of the ovary (Fig. 6).
Several early previtellogenic oocytes, less than 20 µm in
diameter with a distinct nucleolus in the germinal vesicle,
were also found within this same part of the germ zone
(Fig. 7).
Growing oocytes larger than 30 µm in diameter weresurrounded by a thin layer of follicle epithelium. Thesefolliculated oocytes were connected to each germ zone via
their follicles (Fig. 8). Early previtellogenic oocytes larger
than 30 µm in diameter had a distinct nucleolus in their

OVARIAN STRUCTURES IN POLYDESMID DIPLOPODS

Fig. 1

Fig. 2

Fig. 3Fig. 4

Cross-section of adult ovary in Nedyopus tambanus. Dorsal side is oriented towards top of the image. Specimen prepared using hematoxylin andeosin staining. Section shows paired germ zones. Ovarian epithelium of each germ zone forms a shallow and narrow fold that extends towards
the ovarian lumen. Arrows indicate the narrow openings of these folds, which create a pair of longitudinal grooves on the dorsal surface of theovary. ac: alimentary canal, oe: ovarian epithelium, ol: ovarian lumen. Scale = 100 µm.
Cross-section of adult ovary in Nedyopus tambanus. Dorsal side is oriented towards top of the image. Magnification of the right germ zone shown in
Fig. 1. Small arrowheads indicate ovarian epithelium. Arrow indicates a narrow opening of the germ zone fold, which creates a longitudinal grooveon the dorsal surface of the ovary. Large arrowhead indicates position of oogonia. Growing oocytes are arranged in order of increasing volume. epo:
early previtellogenic oocyte, evo: early vitellogenic oocyte, lpo: late previtellogenic oocyte, ol: ovarian lumen. Scale = 50 µm.
Cross-section of adult ovary in Oxidus gracilis. Dorsal side is oriented towards top of the image. Specimen prepared using hematoxylin and eosin
staining. Section shows paired germ zones. Ovarian epithelium of each germ zone forms a shallow and narrow fold that extends towards theovarian lumen. Arrows indicate narrow openings of these folds, which create a pair of longitudinal grooves on the dorsal surface of the ovary. ol:
ovarian lumen, vnc: ventral nerve cord. Scale = 50 µm.
Cross-section of young adult ovary in Oxidus gracilis. Dorsal side is oriented towards top of the image. Specimen prepared using hematoxylin
and eosin staining. Extended part of left germ zone shows growing oocytes arranged in order of increasing volume. Small arrowheads indicate
ovarian epithelium. Arrow indicates a narrow opening of the germ zone fold, which creates a longitudinal groove on the dorsal surface of theovary. Large arrowhead indicates position of oogonia. epo: early previtellogenic oocyte, ol: ovarian lumen. Scale = 10 µm.

78K. YAHATA

large germinal vesicle as well as weakly basophilicooplasm. In late previtellogenic oocytes larger than 80 µm
in diameter, a number of very small lipid droplets appeared
within the ooplasm. These larger oocytes were locatedmore laterally, far from the oogonial area, in each germ
zone.
Eucondylodesmus elegans
The adult ovary in Eucondylodesmus elegans wascomposed of a single median long and sac-like organ,
extending from about the fourth through to the 17th bodysegment. Ovarian epithelium ranging from 1–2 µm inthickness surrounded a wide ovarian lumen (Fig. 9). A pairof germ zones was found in the dorso-lateral region of theovary (Fig. 9). Oogonia with spherical nuclei approximately
4 µm in diameter were found along the medial end of eachgerm zone (Fig. 10). Several growing oocytes larger than
10 µm in diameter were surrounded by a layer of follicle
epithelium. These folliculated oocytes were connected to
the germ zones via their follicles (Fig. 11). Within eachgerm zone, oogonia and several growing oocytes werearranged in order of increasing volume starting at a medial
position and continuing into the lateral region (Figs. 9, 10).
No folded structures were observed around either of theFig. 5

Fig. 6

Fig. 7

Fig. 8germ zones, resulting in the surface of the ovary having asmooth appearance.
All specimens of E. elegans examined in the present
study were young and no information regarding
vitellogenic growth of oocytes could be obtained. Only
approximately 40–50 oocytes in late previtellogenic stagesof oogenetic development were present within each ovary.
Discussion
Comparison of ovarian structure within polydesmid
diplopods
Previous studies (Versluys and Demoll, 1923;
Radhaklishnan and Adiyodi, 1977; Nair, 1981) havedescribed some polydesmid ovaries as “paired (tubular)
ovaries” that connect various sizes of developing oocytes
via their outer surfaces while being surrounded by “single
common ovitube” epithelium that is continuous with theoviductal epithelium. The present study, on the other
hand, noted ovarian epithelia of paired germ zones forminga pair of deep folds that extended toward the ovarian lumen
and created paired longitudinal grooves running along bothsides of the median axis on the outer dorsal surface of theovary. It is possible that previous studies have misidentified
these paired germ zone folds as “paired tubular ovaries”

Cross-section of adult ovary in Epanerchodus orientalis. Dorsal side is oriented towards top of the image. Specimen prepared using hematoxylin
and eosin staining. Section shows a pair of germ zones. Ovarian epithelium of each germ zone forms a deep and wide fold that extends towards
the ovarian lumen. Arrows indicate the narrow openings of these folds, which create a pair of longitudinal grooves on the dorsal surface of theovary. oe: ovarian epithelium, ol: ovarian lumen. Scale = 100 µm.
Cross-section of adult ovary in Epanerchodus orientalis. Dorsal side is oriented towards top of the image. Magnification of the left germ zone
shown in Fig. 5 showing growing oocytes arranged in order of increasing volume. Arrow indicates the narrow opening of the germ zone fold.
Arrowheads indicate position of oogonia. epo: early previtellogenic oocytes, lpo: late previtellogenic oocyte, ol: ovarian lumen. Scale = 100 µm.
Cross-section of adult ovary in Epanerchodus orientalis. Dorsal side is oriented towards top of the image. Extended part of germ zone, including
oogonia and early previtellogenic oocytes, connecting with ovarian epithelium and forming young germ cell-cluster. epo: early previtellogenic
oocyte, oe: ovarian epithelium, og: oogonium, ol: ovarian lumen. Scale = 10 µm.
Cross-section of adult ovary in Epanerchodus orientalis. Dorsal side is oriented towards top of the image. Extended part of germ zone, connecting
early previtellogenic oocyte (epo) and late previtellogenic oocytes (lpo) to the ovarian epithelium (oe) of the germ zone via their own follicle
layers. Arrowheads indicate follicular connections. fc: follicle cell. Scale = 50 µm.

OVARIAN STRUCTURES IN POLYDESMID DIPLOPODS

with a single median sac-like ovary serving as a common“ovitube”.
Kubrakiewicz (1987) observed ovarian structure in
the polydesmid Oxidus gracilis, one of the species
examined in the present study, and illustrated the paired
germ zones as being located in the ventro-lateral parts ofFig. 99the ovary, while failing to mention any folded structurearound them. The present study, however, revealed that O.
gracilis possessed paired germ zones in the dorso-lateralparts of the ovary and that paired germ zone folds extended
toward the ovarian lumen, as seen in other polydesmids.
Results of the present study strongly suggest that

Cross-section of adult ovary in Eucondylodesmus elegans. Dorsal side is oriented towards top of the image. Specimen prepared using hematoxylin
and eosin staining. Section shows a pair of germ zones (arrows). ac: alimentary canal, lpo: late previtellogenic oocyte, oe: ovarian epithelium, ol:
ovarian lumen, vnc: ventral nerve cord. Scale = 50 µm.
Fig. 10 Cross-section of adult ovary in Eucondylodesmus elegans. Dorsal side is oriented towards top of the image. Extended part of left germ zone
showing growing oocytes arranged in order of increasing volume. ac: alimentary canal, epo: early previtellogenic oocyte, fc: follicle cell, ic:
interstitial cell, lpo: late previtellogenic oocyte, oe: ovarian epithelium, og: oogonium, ol: ovarian lumen. Scale = 10 µm.
Fig. 11 Cross-section of adult ovary in Eucondylodesmus elegans. Dorsal side is oriented towards top of the image. Extended part of right germ zone
showing folliculated, early previtellogenic oocyte (epo) connected to the ovarian epithelium (small arrowheads) via its own follicle layer. Large
arrowhead indicates the follicular connection. ac: alimentary canal, fc: follicle cell, lpo: late previtellogenic oocyte. Scale = 10 µm.

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K. YAHATA

Kubrakiewicz (1987) misread the dorso-ventral axis of theovary and failed to notice the folded structures of the germzone epithelium. Kubrakiewicz (1987) observed ovaries
after they had been removed from the bodies of specimens,
possibly allowing the dorso-ventral axis to be misread andthe folded structures to be either partially or entirely
unfastened.
The present study described that the depth and widthof germ zone folds varied among the four polydesmid
species examined. Epanerchodus orientalis exhibited themost developed, deep and wide germ zone folds, while
Eucondylodesmus elegans exhibited no folds around thegerm zones whatsoever. It is possible that the E. elegans
specimens used in the present study were too young toform germ zone folds, and that mature specimens would
have developed these folds; however, young specimens of
the other polydesmid species examined always showedadequately developed folds (e. g., the specimen of Oxidus
gracilis shown in Fig. 4). Alternatively, structuraldifferences in germ zone folds could be related todifferences in fecundity or in the number of eggs per
clutch. E. elegans, whose ovaries lacked germ zone folds,
had only several tens of late previtellogenic oocytes in theadult ovary; conversely, E. orientalis, Nedyopus tambanus,
and O. gracilis had well developed germ zone folds and are
known to lay several hundreds of eggs at once (e. g.,
Causey, 1943; Shinohara, 1957; Murakami, 1962; Komoto
and Matsumoto, 1967). Wider germ zones forming folded
structures likely enable more oocytes to develop
simultaneously.
Comparison with other chilognathan diplopods
The present study identifies several features that canbe used to distinguish polydesmid ovaries from those ofother chilognathans. In polydesmid germ zones, theorderly arrangement of oogonia and growing oocytes
according to their volume was observed. Within each germzone, oogonia and younger oocytes were found within thedorso-medial regions, while larger oocytes were locatedmore laterally. Kubrakiewicz (1987) described a similarorderly arrangement of germ cells in the ovary of Oxidus
gracilis. Based on the results of Kubrakiewicz (1987) andthose of the present study, the orderly arrangement ofgerm cells from the dorso-medial region towards thelateral region according to volume should now berecognized as an ovarian feature common amongpolydesmid diplopods. To ascertain this, future researchshould be undertaken in order to examine chelodesmidean
and dalodesmidean ovaries.
Similar orderly germ cell arrangement has also been
observed in the ovary of Hyleoglomeris japonica, achilognathan species belonging to the order Glomedida(Yahata and Makioka, 1997). However, germ cellarrangement in the ovary of H. japonica clearly differsfrom arrangements observed in polydesmid ovaries. In H.
japonica, oogonia and smaller oocytes are located at thedorsal and ventral edges of each germ zone, while larger
oocytes are located more laterally (Yahata and Makioka,
1997). Phylogenetically, the Glomerida and the
Polydesmida are distantly related to one another (Enghoff,1984), and similar ordered arrangements of germ cells
have not been observed in other diplopod orders.
Therefore, the orderly germ cell arrangements observedin glomerid and polydesmid ovaries likely do not share acommon evolutionary origin.
Acknowledgments: I would like to thank Professor T.
Makioka (University of Tsukuba) for helpful discussion
and constant encouragement throughout this study. I am
indebted to Professor K. Ishii (Dokkyo University Schoolof Medicine) for his kind identification of specimens. I
would also like to thank two anonymous reviewers fortheir helpful comments.
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