Agr. Nat. Resour.

54 (2020) 211–216

AGRICULTURE AND
NATURAL RESOURCES
Journal homepage: http://anres.kasetsart.org

Research article

The ovarian structure and oogenesis of the pea crab Pinnotheres cyclinus

Gordon, 1932: A histological investigation
Sinlapachai Senarata, Lamai Thongboonb, Jes Kettratadc,d, Wannee Jiraungkoorskule,*, Natthakitt To-ornf,
Chanyut Sudtongkonga, Koraon Wongkamhaengg, Mari Carmen Uribeh
a
Department of Marine Science and Environment, Faculty of Science and Fisheries Technology, Rajamangala University of Technology Srivijaya,
Trang Campus, Sikao, Trang 92150, Thailand.
b
Department of Biology, Faculty of Science, Prince of Songkla University, Songkhla 90110, Thailand.
c
Department of Marine Science, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand.
d
Aquatic Resources Research Institute, Marine Ecology and Marine Resources Utilization Research Unit, Chulalongkorn University, Bangkok
10330, Thailand.
e
Pathology Information and Learning Center, Department of Pathobiology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand.
f
Program of Fisheries Science, Faculty of Agricultural Technology and Agro-Industry, Rajamangala University of Technology Suvarnabhumi,
Ayutthaya 13000, Thailand.
g
Department of Zoology, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand.
h
Laboratorio de Biologıa de la Reproduccion Animal, Departamento de Biologıa Comparada, Facultad de Ciencias, Universidad
Nacional Autonoma de Mexico, 04510 Mexico D.F., Mexico.

Article Info Abstract

Article history: The pea crab Pinnotheres cyclinus is a parasite of cultured mollusks. However, the information on
Received 6 November 2017
reproductive histology of this crab has remained limited. Therefore, the present study investigated
Revised 14 August 2019
Accepted 19 August 2019 the ovarian structure and oogenesis of Pinnotheres cyclinus during maturation using histological
Avariable online 24 April 2020 techniques. Histologically, the ovary of this crab was found to be surrounded by a thin epithelium
and connective tissue of the ovarian wall. Different phases of oogenesis were observed in the
Keywords:
germinal area and could be classified into four phases: oogonial proliferation, the primary growth
Histology, phase, the secondary growth phase, and the atretic oocyte phase. An oogonium was located in the
Microanatomy, ovarian cyst of the ovarian lobe, which was surrounded by a layer of pre-follicular cells. During
Oocyte,
the primary growth phase, oogonia continued to develop in the ovarian cyst, accumulating lipids
Ovary,
Thailand and cortical alveoli. The appearance of spherical yolk granules related to changes in follicular cells during
the secondary growth phase was also observed. These yolk granules reacted positively to Masson’s
trichrome and Mollary’s trichrome staining, implying the presence of mucopolysaccharides and
glycoproteins. Atretic oocytes were also found. Stages of embryonic development were also
observed, including the formation of egg membranes covering embryos. Consequently, a fertilized
egg was then filled with yolk granules, which all gradually combine to become one within the egg.

* Corresponding author.
E-mail address: [email protected] (W. Jiraungkoorskul)

online 2452-316X print 2468-1458/Copyright © 2020. This is an open access article, production and hosting by Kasetsart University of Research and Development institute on
behalf of Kasetsart University.

https://doi.org/10.34044/j.anres.2020.54.2.13
212 S.Senarat et al. / Agr. Nat. Resour. 54 (2020) 211–216

Introduction et al., 2018). These sections were dehydrated using a series of

alcohols from 95% ethyl alcohol to absolute alcohol and embedded in
Pinnotheres cyclinus, locally known as the pea crab, is a symbiont paraffin. Each serial section was cut to a thickness of 4 µm and then
and parasite of bivalves such as scallops and mussels (Silas and stained with hematoxylin-eosin (H&E) to study the basic structure of
Alagarswawi, 1967; Majchacheep, 1989). This species is broadly the ovarian tissue. For the detection of chemical details, some sections
known in China, Japan, Korea, and other Southeast Asian countries were histochemically stained with Masson’s trichrome (MT), periodic
(Miyake et al., 1962; Silas and Alagarswawi, 1967; Morton and Morton, acid-Schiff (PAS), or cresyl violet (CV) (Presnell and Schreibman,
1983). This symbiotic/parasitic crab is of interest in mariculture as it 1997; Suvarna et al., 2018). The ovarian structure and oogenesis
positively or negatively affects the life cycle of its bivalve host, which processes of P. cyclinus were examined and photographed with
is cultivated and/or harvested from wild populations for commercial a Leica TE2000-U, following guidelines described by Mota and
exploits (Majchacheep, 1989; Sun et al., 2006; Science Learning Hub, Tomé (1965), Kulkarni et al. (1991), Becker et al. (2011), and Ravi et al.
2019). To date, P. cyclinus controlling in several areas has shown little (2013). Oocyte and follicular layer diameters were measured using
success (Majchacheep, 1989; Science Learning Hub, 2019). a calibrated micrometer with a Leica TE2000-U. A schematic diagram
Comprehensive data concerning the ovarian and oocyte structure of the ovarian structure and the development stages of oocytes in this
development of P. cyclinus is an important knowledge for further species was made using Adobe Illustrator (version CS6).
understanding on reproductive biology, reproductive cycle and
spawning season of the crab (Castiglioni et al., 2007; Stewart et al., Results and Discussion
2007). During oocyte development within crabs, oogonia are produced
in the perennial germinal zone and divide mitotically to form primary Histological observation of the ovarian structure
oocytes (Stewart et al., 2007). Multiple stages of oocyte differentiation
can be classified according to morphological and histological The results showed that all specimens had attained ovarian
characteristics. Five stages of oocytes differentiation were found maturation, at which point the ovarian structures were located among
in Scylla serrata, S. paramamosain, and Portunus pelagicus (Stewart the digestive organs (Figs. 1A) and surrounded by digestive glands (Fig.
et al., 2007; Islam et al., 2010). Subsequently, Castiglioni et al. (2007) 1B). The ovarian wall of P. cyclinus was histologically composed of
proposed that the oogenetic process of the crab Uca rapax consists a thin layer of epithelium and connective tissue (Figs. 1C–D). However,
of six stages. In Travancoriana schirnerae, oocyte development was it differed from the ovarian walls of other decapods, which consist of
categorized into ten stages (Smija and Devi, 2015). multiple layers: an outermost thin pavement epithelium, a middle layer
The knowledge of P. cyclinus  reproductive cycles of  is necessary of connective tissue, and an innermost layer of germinal epithelium,
to control the population of this crab and maintain the economic as was reported in Portunus pelagicus (Ravi et al., 2013), Litopenaeus
reserves of economically valuable mollusks. However, information setiferus (King, 1948), Monodon monoceros (Abraham, 2005),
regarding the reproductive histology of this crab is lacking. In and Ranina ranina (Minagawa et al., 1993). In P. cyclinus, the layers
the present work, we describe in detail the ovarian structure and of the ovarian wall were filled with interstitial tissue, which had
oogenesis process of P. cyclinus using histological and histochemical separated into several ovarian lobes (Figs. 1C–D). Each lobe composed
techniques. The dynamics of these oogenetic processes were also of several ovarian cysts (Figs. 1D and 2). The different stages of oocyte
schematically drawn. Data drawn from our observations can help development occurred in the ovarian cysts (Fig. 2). The following
to increase understanding of the female reproductive biology of  four phases of oocyte development were classified based on cell
P. cyclinus. This knowledge can be used to respond to the infective size, nucleus characterization, cytoplasmic properties, and degree
effects of this crab on bivalve mariculture and other aquatic organisms of yolk granules: oogonia proliferation, the primary growth phase
both in Thailand and elsewhere. (PGP), the secondary growth (SG) phase, and the atretic oocyte phase.
These criteria have been commonly used to classify oogenesis in 
Materials and Methods P. pisum (Becker et al., 2011) and other decapods (Mota and Tomé,
1965; Kulkarni et al., 1991; Ravi et al., 2013).
Ten female P. cyclinus with a mean carapace width of 30.50 ±
0.45 mm (CW range: 28.0–31.0 mm) were collected from infected  Histological observation of oogenesis
Perna viridis from the Upper Gulf of Thailand in  Chonburi Province,
near Si Racha District (13°10’28.4”N, 100°55’10.1”E), from Oogonia proliferation (Op) 
December 2015 to February 2016. Following capture, all live crabs The initial stage of oogonial (Og) maturation was initiated in the
were air-lifted and transported to a laboratory. The crabs were cell nest of the internal germinal area (Figs. 3A–B). Each cell nest
euthanized by rapid cooling shock (Wilson et al., 2009), and then the was separated from other oocyte stages by the basement membrane
dorsal parts of their carapaces were opened. Then, the whole body (Fig. 3A). This observation is consistent with previous observations
of each crab, without the carapace, was fixed in Davidson’s fixative (e.g., Ryan, 1967; Eurenius, 1973), such as observations of Libinia
(48 hr) and processed into thin sections according to standard emarginata (Hinsch and Cone, 1969) and R. ranina (Minagawa et al.,
histological techniques (Presnell and Schreibman, 1997; Suvarna 1993). Each individual oogonium had an oval to spherical shape.
 S.Senarat et al. / Agr. Nat. Resour. 54 (2020) 211–216 213

Primary growth phase (PGP)

In this phase, we classified two stages of oocyte development:
(1) perinucleolar (Pn) oocytes and (2) oil droplets and cortical alveolar
(Oc) oocytes (Figs. 3B-E), which were still present in germinal cysts
as follows. The Pn oocytes had spherical shapes and were larger
than oogonia, approximately 40 µm in diameter (Fig. 3B). Each Pn
oocyte had a central nucleus with a mean diameter of 20.00±0.56 µm
containing euchromatin and small blocks of heterochromatin, which
were pale-stained and distributed throughout the nucleoplasm
(Fig. 3B). At the same time, multiple nucleoli with a mean diameter
of 5.00±0.92 µm were located along the nuclear membrane (Fig. 3D).
The ooplasm exhibited strong basophilic staining (Figs. 3B and C),
and the basophilic characterization of each oocyte was previously
related to an accumulation of ribosomes and, for a period, an intense
vesicular nucleus (Beninger et al., 1993) with three to four chromatin
clumps (Weitzman, 1966; Okumura and Sakiyama, 2004). Similar
to those of two other crab species - Cambarus virilis (Beams and
Fig. 1. Light photomicrograph of the ovarian parenchyma of Pinnotheres cyclinus.
A-B: The localization of the ovarian structure (Ov) among the digestive organ
Kessel, 1962) and Cancer pagurus (Eurenius, 1973) - each Pn oocyte
(Do) consisting of several digestive glands (Dg) was observed. C-D: The ovarian contained an enrichment of ribosomes and the development of rough
structure was surrouned with ovarian wall (Ow); the ovary comprised of endoplasmic reticulum as revealed by ultrastructural examination.
several ovarian lobes (Ol). Note: Is = interstitial tissue, Ocy = Ovarian cysts. During the process of folliculogenesis, follicular cells became
A-B, D = Masson’s Trichrome (MT), C = haematoxylin–eosin (H&E). completely surrounded by Pn oocytes (Fig. 3D).
The oil droplets and Oc oocytes had ovoid shapes and had
slightly increased in size to a diameter of approximately 70 µm,
but a decrease in nuclear diameter (of approximately 25 µm) was
recorded as well. The ooplasm of this type of oocyte was deeply
stained with hematoxylin (Fig. 3B). Interestingly, Oc oocytes
increased in cell size due to an accumulation of vacuolated globules,
such as oil droplets and cortical alveoli (Fig. 3E). The appearance
of oil droplets was visible as clear vacuoles because the oil droplets
dissolved during histological preparation (Figs. 3D–E). This pattern
of oil droplet presentation has been commonly recorded in decapods
(Chen et al., 2004; Walker et al., 2006). Lipid accumulations have
been reported as nutrient reservoirs for nutritionally dependent zoeae
(Chen et al., 2004; Walker et al., 2006). The presently observed cortical
alveoli were slightly colored by Haemotoxylin and Eosin (H&E
staining) (Fig. 3E) and positively colored with PAS staining (data not
shown), indicating the presence of glycoproteins. This finding could
indicate a build-up of cortical rods, which function in forming a jelly
layer to cover an oocyte (Clark et al., 1990). Supporting follicular
Fig. 2. Light photomicrograph of the ovarian structure of Pinnotheres cells were well-defined and approximately 2 µm in diameter (Fig. 3D).
cyclinus. Each ovarian lobe (Ol) contained various ovarian cysts (Ocy) which Comparable situations have been investigated in several decapods,
were consisted of different oocytes. A = Masson’s Trichrome (MT). such as Penaeus semisulcatus, P. monodon (Ongvarrasopone et al., 
2006),  Marsupenaeus japonicus,  and other penaeoid shrimps
The oogonium had a mean diameter of 10±0.98 µm, whereas the (Okumura et al., 2006).
average nuclear diameter was approximately 3.4±1.03 μm (Figs. 3A
and C). A slightly oval nucleus occurred and contained moderately Secondary growth (SG) phase 
condensed heterochromatin, which was arranged as a clock-face In this phase, the following three stages of SG were classified:
feature (Figs. 3A–B). A spherical nucleolus was also observed the early SG stage (Esg), the late SG stage (Lsg), and the fully grown
(Fig. 3A). The nucleus was partially surrounded by slightly basophilic oocyte stage (Fgo), which remained developed in the ovarian cysts
cytoplasm (Fig. 3A). During the process of folliculogenesis, which was (Figs. 3C–E and 4).
observed using MT and CV staining methods, each cell was associated
with a few squamous-shaped prefollicular cells (Figs. 3A–B).
214 S.Senarat et al. / Agr. Nat. Resour. 54 (2020) 211–216

During the Esg step, the oocytes suddenly increased in size to Lsg was accompanied by a rapid increase in cell volume, up to 200 µm
approximately 70 µm in diameter due to an accumulation of yolk in diameter, due to accumulated yolk granules (Figs. 4A–C). The fusion
granules (Figs. 3C–D). Spherical small yolk granules were detected by of the yolk granules or yolk plate, with a maximum diameter of 15 µm,
their deep reddish stains following their positive reactions to MT staining, was initially observed in eosinophilic ooplasm (H&E staining; Fig. 4A)
implying the presence of glycoproteins and mucopolysaccharides and reddish ooplasm (MT staining; Fig. 4B), but these yolk granules
(Figs. 3C–D); similar results were previously observed in  Malacostraca did not react to the CV staining method (Fig. 4C). Some yolk granules
(Charniaux-Cotton, 1975), Gecarcinus lateralis (Weitzman, 1966),  were also hydrated and coalesced. At this stage, the irregular nuclei
Chionoecetes opilio (Beninger et al., 1993), Acanthomysis robusta  were reduced in size and number and had moved to the animal pole
(Okumura, 2003), and Portunus trituberculatas (Hamasaki et al., 2004). of the oocyte through germinal vesicle migration (GVM). At the
During oocyte differentiation, an irregular outline of the center nuclear same time, the nucleoli were not visible. A reduced follicular cell was
structure was slightly displaced toward the animal pole (Stewart et al., observed (Figs. 4A–D), and at the end of this stage, small nuclei were
2007). During Esg, the oocyte was more visible and still surrounded by observed (Figs. 4A–D).
a single layer of supporting follicular cells; in contrast, this layer also Fgo consisted of fully mature oocytes. Oocytes were largest
exhibited increasing size/density/width (Figs. 3D–C). These changes at this stage, with maximum diameters ranging from 250–300 µm.
in oocyte structure resulted from follicular changes during this stage Due to membrane breakdown, also called germinal vesicle breakdown,
and were considered to have been responsible for heterosynthetic yolk no nucleus was detected during this stage (Figs. 4E–F). The oocyte
deposition in our specimens, as has been described previously by Islam still exhibited an increased abundance of yolk plates in the ooplasm.
et al. (2010) and Yano (1988) in brachyurans and other crustaceans.
Atretic oocyte phase
Although most post-ovulatory phases were not observed throughout
the present study, the atretic oocyte phase was commonly found among
the oocytes. Atretic oocytes during the PGP, the irregular nucleic
shapes were surrounded with the detachment of the thin follicular

Fig. 3. Light photomicrograph of the oogenesis of Pinnotheres cyclinus.

A: Oogonial proliferation containing the oogonium (Og). B-E: Different
stages of oocytes including perinucleolar oocyte (Pn), oil droplets and cortical
alveolar oocyte (Oc), Full-grown oocyte step (Fgo) and late secondary growth
step (Lsg) throughout atretic oocyte of previtellogenic stage (Ap). Note:
Ag = acidophilic granulocytes, Bm = basement membrane, Ca = cortical alveoli, Fig. 4. Light photomicrograph of the oogenesis of Pinnotheres cyclinus.
Cf = clock-face feature, Esg = early secondary growth step, Fc = follicular cell, A-D: Late secondary growth step (Lsg) containing the large yolk granules
He = heterochromatin, Ld = lipid droplet, Nu = nucleus, Nuc = nucleolus, (Yg). E-F:The absence of nucleus was found in full-grown oocyte step (Fgo).
Spc = simple pre-follicular cell, Yg = yolk granules. A, C-D = Masson’s Note: Fc = follicular cell, Nu = nucleus. A = haematoxylin–eosin (H&E),
Trichrome (MT), B = cresyl violet (CV), E = haematoxylin–eosin (H&E). B, D, E = Masson’s Trichrome (MT), C, F = cresyl violet (CV).
 S.Senarat et al. / Agr. Nat. Resour. 54 (2020) 211–216 215

layer (MT staining; Fig. 3D). It could be argued that the cause of In the present study, the application of histological techniques
pronounced atresia in aquatic organisms is related to a wide range of clearly revealed the ovarian structure and oogenctic processes of 
environmental stressors such as heavy metals (Pierron et al., 2008), P. cyclinus for the first time, as summarized in Fig. 6. This study
endocrine-disrupting chemicals (Pollino et al., 2007), starvation, and provides basic information that may help in the study of the
lipid-poor diets (Hunter and Macewicz, 1985). Additionally, it should reproductive cycle and reproductive physiology of P. cyclinus.
be noted that in crabs and crustacea, oocytes and ovarian lobes begin
to degrade at the end of ovarian development and, subsequently,
germinal areas appear for the next cycle (Subramoniam, 2016).
Alternatively, this unusual progression of atresia may be associated
with the unusual habitat/lifestyle of this crab and/or its unusually high
fecundity.

Histological observation of embryonic development

During our observation of the ovarian structure of the pea crab,
its embryonic stages were also detected (Figs. 5A–F). First, the
embryonic surface was covered by the egg membrane, referred to as
the vitelline envelope. The embryonic surface also reacted positively
to MT and CV staining. Subsequently, the fertilized egg was filled
with yolk granules, which were homogeneously distributed within the
egg. This stage might have occurred approximately three days after
fertilization, as was previously observed in other decapod species Fig. 6. Schematic diagram of the ovarian structure and oogenesis of
(Habashy et al., 2012). Pinnotheres cyclinus. The ovarian lobe (Ol) was contained with several ovarian
cysts (ocy), which each ocarian cyst consisted of the differentiating stages of
oocyte including oogonium (Og), perinucleolar stage (Pn), oil droplets and
cortical alveolar step (Oc), early secondary growth step (Esg), late secondary
growth step (Lsg) and full-grown oocyte step (Fgo). Note: Ca = cortical alveoli,
Fc = follicular cell, Ld = lipid droplet, Nu = nucleus, Ocy = ovarian cyst, Ol =
ovarian lobe, Yg = yolk granule

Conflict of Interest

There is no conflict of interest.

Acknowledgement

This research was supported by The 100 th Anniversary

Chulalongkorn University Fund for Doctoral Scholarship. Special
thanks to language editing service provided by KU Research and
Development Institute, Kasetsart University.

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