Structure of the male reproductive system of the blue ...

Acta Zoologica (Stockholm)

doi: 10.1111/azo.12017

Structure of the male reproductive system of the blue swimmer crab Portunus pelagicus (Decapoda: Portunidae) Raghunath Ravi, Mary Kurian Manisseri and Nandiath Karayi Sanil

Abstract Central Marine Fisheries Research Institute, Post Box No. 1603, Kochi, 682018, India Keywords: Portunus pelagicus, maturation, gonoduct, testis, vas deferens Accepted for publication: 09 November 2010

Raghunath, R., Manisseri, M. K. and Sanil, N. K. 2012. Structure of the male reproductive system of the blue swimmer crab Portunus pelagicus (Decapoda: Portunidae). — Acta Zoologica (Stockholm) 00: 000–000. An attempt was made here to study the structure of the male reproductive system of Portunus pelagicus, which would improve the knowledge base on the reproductive biology of the species and also help in the maintenance of broodstock under controlled conditions. Male P. pelagicus of different sizes were collected from the Palk Bay off Mandapam (9°17′ N, 79°9′ E) and maintained under controlled conditions for the study. Tissues from testis, anterior vas deferens (AVD), median vas deferens (MVD), posterior vas deferens (PVD), ejaculatory duct and penis were fixed in Bouin’s fluid and 2.5% buffered glutaraldehyde separately and processed for light and electron microscopic studies, respectively. The reproductive system consisted of testis, commissure, vas deferens, ejaculatory duct and penis. The vas deferens was divided based on the morphology and/or histology into AVD, MVD and PVD. The AVD was further divided based on histology into proximal and distal regions, and the MVD, based on diameter into major and minor coils. The testicular lobe had several lobules with a central seminiferous tubule, which continued till the penis. The seminiferous tubule was lined by a layer of cuboidal or columnar epithelium. The lining of the central tubule of the vas deferens formed several ‘folds’, which at times formed ‘pouches’. High incidence of cell organelles in the columnar epithelial cells, aggregations of vesicles and occurrence of blebs at the luminal periphery and the projection of numerous microvilli containing electron-dense materials into the lumen from the cell lining denoted high secretory activity of the epithelial cells. Raghunath Ravi, Marine Biodiversity Division, Central Marine Fisheries Research Institute, Post Box No. 1603, Kochi 682 018, Kerala, India. E-mail: [email protected]

Introduction The marine blue swimming crab Portunus pelagicus is a candidate species for culture because of its fast growth, attractive appearance and taste. The species is available throughout the coast of India especially in the south-east and the south-west region and breeds round the year (Pillai and Nair 1973). Because commercial hatcheries do not exist for the species (Soundarapandian and Tamizhazhagan 2009), the farmers are forced to depend on the wild for juvenile crabs for culture practices. Difficulties in obtaining juveniles from the wild, their non-uniform size and concerns of stock depletion due to © 2012 The Authors Acta Zoologica © 2012 The Royal Swedish Academy of Sciences

over exploitation have encouraged research activities to frame out hatchery technology of the species. Production of seeds on a large scale is also important in formulating conservation management strategies. Sea ranching of crab juveniles is a good method to enhance the natural stock. Studies on the male reproductive morphology, anatomy and spermatophore formation help in understanding the process of sexual maturation and the changes taking place in the reproductive cycle, which are prerequisites to attempt spermatophore preservation for hatchery and aquaculture purposes. Anatomical and histological study of the male reproductive system has been carried out in many crabs (e.g. the blue crab

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Male reproductive system of Portunus pelagicus Ravi et al.

Callinectes sapidus (Cronin 1947), marine swimmer crab Portunus sanguinolentus (Ryan 1967), snow crab Chionoecetes opilio (Sainte-Marie and Sainte-Marie 1999), deep water crab Charybdis smithii (Balasubramanian and Suseelan 2000), Jonah crab Cancer borealis (Moriyasu et al. 2002), red-clawed mangrove tree crab Goniopsis cruentata (Garcia and Silva 2006), mangrove land crab Ucides cordatus (Castilho et al. 2007) and freshwater crab Barytelphusa cunicularis (Sherkhane et al. 2010)). In C. smithii, the testis is a tubular organ with a double-layered wall and is composed of numerous acini or follicles, which are arranged around a central seminiferous duct (Balasubramanian and Suseelan 2000). In C. borealis, the vas deferens is divided into two: proximal region, which is the site of the formation and storage of fully formed spermatophores and seminal fluid, and distal region, which is the site of the formation of the sperm plug components (Moriyasu et al. 2002), whereas the vas deferens is divided into three parts in U. cordatus (Castilho et al. 2007), four parts in the Spanish lobster Scyllarus chacei (Hinsch and McKnight 1988) and eight parts in the small hermit crab Diogenes pugilator (Manj on-Cabeza and Raso 2000). Sperm development in decapod crustaceans may be synchronous (only one developmental stage in one testicular lobule) or asynchronous (more than one developmental stage in one testicular lobule) depending on species (Krol et al. 1992). In crustaceans, the sperm masses are surrounded by the layers of acellular secretions produced by the vas deferens forming spermatophores (Aiken and Waddy 1980). Several authors have carried out ultrastructural studies on the testis and vas deferens of crabs (e.g. Langreth 1969; Hinsch and Walker 1974; Payen 1977; El-Sherief 1991; Klaus et al. 2011). The present work attempts to reveal the microscopical and ultrastructural details of the different parts of the male reproductive system of P. pelagicus. Materials and methods

Acta Zoologica (Stockholm) 0: 1–10 (November 2012)

cessed to thin sections (6–8 lm) (Bell and Lightner 1988), stained and counter-stained with Delafield’s haematoxylin and 1% alcoholic eosin, respectively. The sections were mounted on slides with DPX and examined under microscope (Olympus CX 41, USA). Based on gonadal morphology and histology, animals were divided into immature and mature, following Ryan (1967). Ultrastructural study For the ultrastructural study using transmission electron microscopy (TEM), tissues from different regions of the male reproductive organs of animals under immature and mature stages were processed to ultrathin sections (Dawes 1988). Tissues of 1 mm3 size were cut from different regions such as testis, AVD, MVD, PVD, ejaculatory duct and penis, fixed in 2.5% buffered glutaraldehyde, washed with sodium cacodylate buffer (0.1 M, pH 7.2) and kept overnight in fresh buffer. The washed tissues were post-fixed in 1% osmium tetroxide, washed with sodium cacodylate buffer and dehydrated in ascending concentrations of acetone. The tissues were infiltered with Spurr’s low viscosity resin (Spurr 1969), embedded in plastic vials and polymerized at 70°C. Ultrathin sections of 60–90 nm were cut using ultramicrotome (LKB-Nova, Austria), double-stained with uranyl acetate and lead citrate (Reynolds 1963), mounted on grids, observed and photographed under transmission electron microscope (Hitachi H-600, Japan) at 75 KV. Results In immature animals, the testis, which appeared as a thin translucent thread, was hardly visible. The remaining portions were very difficult to distinguish. Histologically, different parts of the immature testis had only gonial cells or were empty. In mature animals, all the parts were obvious, and the sperm cells were at different stages of development.

Animal collection and maintenance Twelve male P. pelagicus of carapace width range from 62 mm to 133 mm (70–151 g) were collected monthly from Palk Bay off Mandapam, (9°17′ N, 79°9′ E) during June 2007 – January 2008. The animals were transported live to the wet laboratory and maintained in 1-ton fiberglass reinforced plastic (FRP) tanks with continuous aeration for the study. Feeding was carried out with raw squid (Sepia pharaonis) and clam (Meritrix meritrix) meat (1 : 1) ad libitum daily. Water exchange (70%) and feed waste removal were carried out every day. Light microscopic study Small pieces of tissues from different regions of testis, anterior vas deferens (AVD), median vas deferens (MVD), posterior vas deferens (PVD), ejaculatory duct and penis were pro-

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Gross morphology of male gonoducts The testis, located in the cephalothorax, was a complex, pale white, tubular organ with many lobules (acini) arranged around a central seminiferous tubule that ran through the entire stretch of the testis. It was connected to the vas deferens that, based on the morphological and histological studies, was divided into AVD, MVD and PVD (Fig. 1), following Cronin (1947). These portions were further divided into different regions as per Ryan (1967). The AVD was a delicate, translucent and highly coiled ball-like structure. Due to extreme coiling, the histological sections always had several loops, and any attempt to uncurl caused rupture. The next part was the loosely coiled MVD, which was pure white and opaque due to the presence of white granular fluid surrounding the sperm mass inside. The last portion of the vas deferens was the colourless PVD. Although the diameter of the MVD and the © 2012 The Authors Acta Zoologica © 2012 The Royal Swedish Academy of Sciences

Ravi et al. Male reproductive system of Portunus pelagicus


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Fig. 1—Drawing of the male reproductive system of Portunus pelagicus showing testis (T), anterior vas deferens (AVD), median vas deferens (MVD), posterior vas deferens (PVD), ejaculatory duct (ED) and penis (P).

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minor coil of the PVD were the same at the beginning of the latter, they were easily distinguishable from one another. PVD gradually tapered to join the tubular ejaculatory duct, which continued downward and opened at the base of the fifth pair of walking leg on the eighth thoracic segment in the form of a penile papilla/penis (Fig. 1). The penis was a flaccid white tubular organ that assisted the transfer of male sexual contents from the ejaculatory duct to the grooves of the first pair of abdominal appendages (gonopods) during copulation. The accessory reproductive structure comprised of two pairs of abdominal appendages (pleopods), which were modified structurally for copulatory function.

Fig. 2—Cross section of testicular lobe. — A. Showing several lobules

(Lo) with spermatogonial cells (SG), spermatocytes (SC), spermatozoa (SZ), seminiferous tubule (ST) and haemal sinuses (HS). The membraneous wall (MW) of the lobule and nuclei of the sperm cells (arrowheads) are also seen. — B. Seminiferous tubule (ST) showing spermatozoa (SZ) and columnar cell (Co) lining. — C. Higher magnification of testicular lobules showing membraneous walls (arrow heads). Scale bars: A and B = 50 lm, C = 100 lm.

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Histology and ultrastructure Testis. The testicular lobes were partially or fully divided into several lobules/acini delimited by a thin membrane, which was slightly stained by eosin (Fig. 2A,C). Each lobule comprised of sperm cells in uniform developmental stage (synchronous type) (Fig. 3A–C). The lobules of the testis were arranged around an unbranched collecting duct called seminiferous tubule, lined by columnar epithelial cells (Fig. 2B). More than one seminiferous tubule was seen in sections due to the looping of the testis and the tubules were filled with spermatozoa. The lobules had either direct access to the seminiferous tubule or were connected indirectly through other lobules. The entire testis was covered by a double-layered wall, made up of an outer fibrous connective tissue layer and an inner delicate membraneous layer. Basophilic haemal sinuses were found in between the lobules, which were invariably bathed in eosinophilic haemolymph (Fig. 2A). The germinal zone was formed by the aggregation of spermatogonial cells at the periphery of the lobules (Fig. 3A,B). These cells had very large unstained vesicular nuclei with basophilic nucleoli, and the cytoplasm was just a thin slightly basophilic band around the nucleus (Fig. 4). Somatic cells called nutritive cells/nurse cells were found dispersed in between the spermatogonia. The spermatocytes and spermatozoids had condensed basophilic nuclei with slightly eosinophilic © 2012 The Authors Acta Zoologica © 2012 The Royal Swedish Academy of Sciences

B

C

Fig. 3—Testicular lobule showing sperm cells under different stages

of development. — A. Synchronous development of sperm cells. Lobules filled with spermatogonial cells (SG) forming germinal zones (GZ) and/or spermatids (SD) are seen. Arrowhead shows the nuclei of sperm cells. — B. Higher magnification of spermatogonial cells with nucleus (N). — C. Higher magnification of spermatids with nucleus (N). Scale bars: A = 100 lm, B & C = 25 lm.

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Fig. 5—Three-layered wall of the anterior vas deferens (AVD) show-

ing exterior connective tissue layer (Ct), middle muscle layer (MS) and inner columnar cell layer (Co). Scale bar = 4 lm.

Fig. 4—Testicular acinus showing spermatogonial cell (SG) and an

adjacent nurse cell NC). The respective nuclei (N) and cytoplasm (Cy) are visible. Arrowhead shows heterochromatin granules in the nucleus of spermatogonia. Scale bar = 4 lm.

similar to those in the proximal portion except that the cuboidal cells of the epithelial lining were replaced by tall columnar cells with strongly basophilic lobated nuclei and

cytoplasm. The spermatogonial cells developed into spermatozoa as they moved towards the collecting duct. The size of the germ cells decreased from gonial cells to spermatids and then increased. In immature animals, the lobules of the testis contained only a narrow germinal zone with non-differentiated germ cells. Anterior vas deferens. This was a highly coiled compact mass held together by connective tissue. Although it was not possible to subdivide it based on morphology, differences in histological sections helped its segregation into proximal and distal portions. The proximal portion of the AVD had a rough circular to oval shape and constituted a duct/lumen towards the middle. The lumen was lined with cuboidal epithelial cells, which was covered by a layer of muscle tissue, and the outermost layer was formed by a connective tissue sheath. The cuboidal epithelial cells had circular to oval-shaped basophilic nuclei and weakly eosinophilic cytoplasm. The epithelial lining of the lumen had a few inward folding, where the cells became columnar with basophilic nuclei (Fig. 5). A layer of basophilic heterogeneous granular matrix was found along the luminal periphery and also within the folds. Another layer of homogeneous eosinophilic matrix filled the lumen. Sperm masses were seldom noticed in the sections studied. In cross section, the distal portion of the AVD had a rough ‘U’ shape (Fig. 6). The layers covering the duct were

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Fig. 6—Distal anterior vas deferens showing sperm mass (SM), sper-

matophoric matrix (spm), folds (F) and columnar cells (Co) with nuclei (arrowheads). Inset shows columnar cells in higher magnification. Scale bar = 100 lm. © 2012 The Authors Acta Zoologica © 2012 The Royal Swedish Academy of Sciences


eosinophilic cytoplasm. The epithelial lining of the lumen showed less folding with respect to that in the proximal portion. A few intensely stained nucleoli were also observed in the nuclei of the columnar cells. Aggregations of tiny secretory vesicles and blebs formed by the columnar cell membrane were observed at the apical region of the epithelial cells (Figs 7 and 8). The epithelial columnar cells showed increased prominence in cell organelles such as Golgi apparatus, rough endoplasmic reticulum and mitochondria near the epithelial lining. Numerous microvilli containing electron-dense materials projected into the lumen from the cell lining (Fig. 7). The vesicular components released by the columnar cell lining gave a frothy appearance to the periphery of the lumen, and masses of sperm cells were generally seen towards the centre of the lumen (Fig. 6). Two types of matrices were observed in the AVD, homogeneous sperm matrix and electron-dense, flocculent and globular spermatophoric matrix (Fig. 9). The basophilic masses of spermatozoa were embedded in the former and the spermatophores in the latter (Fig. 6 and 9). The spaces between the loops of the AVD were filled with haemal sinuses, haemolymph and connective tissue. In immature animals, the AVD was a simple tube with an outermost connective tissue sheath and an inner circular muscle layer with a few blood vessels in between. The lumen consisted of undifferentiated sper-


Fig. 8—Cell membrane (cm) of the columnar cell (Co) lining of ante-

rior vas deferens (AVD), actively participating in the transfer of materials from the cell to the lumen (LU) by the process of ‘blebbing’. Blebs (arrowheads) formed by cell membrane are visible. Scale bar = 2 lm.

Fig. 9—Spermatophore in the distal anterior vas deferens. Spermato-

zoa (sz), sperm matrix (sm), spermatophoric wall (sw) and spermatophoric matrix (spm) are seen. Scale bar = 4 lm.

matogonial cells with large nucleus containing diffused chromatin. Fig. 7—Luminal periphery of anterior vas deferens (AVD), showing

epithelial lining of lumen with secretory vesicles (v) and microvilli (Mv). Scale bar = 0.5 lm. © 2012 The Authors Acta Zoologica © 2012 The Royal Swedish Academy of Sciences

Median vas deferens. Based on the diameter, MVD was divided into major coil (broader) and minor coil (narrower),

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but the histological sections did not show any difference in the internal structure. The general structure was similar to that of the AVD. The lumen wall had three layers viz. inner columnar epithelial cell layer, middle muscular layer and an outer connective tissue layer. In the major coil, the columnar epithelium predominantly had basal nuclei; medial and apical nuclei were also observed. The nuclei were strongly basophilic, and the cytoplasm was faintly eosinophilic. The epithelial lining was characterized by several folds as in the AVD, but more in number, forming pouches, which were filled with eosinophilic homogeneous secretion, probably secreted by the columnar epithelial lining (Fig. 10). The lumen also was filled with the same secretion. The luminal periphery of the epithelial cells actively participated in secretion, which was evidenced by the presence of numerous microvilli projecting into the lumen, array of rough endoplasmic reticula, vesicles released from the columnar cells and electron-dense bodies at the boundary between lumen and the epithelial lining (Fig. 11). In certain columnar cells, the nuclei were lobated with deeply stained nucleoli (Fig. 12). The lumen comprised of roughly circular spermatophores bathed in spermatophoric matrix (Fig. 10). The number of pouches formed by folding of the epithelial lining was much more in the minor coil than that in the major coil, which resulted in its contraction and deformation. There was also an increased prominence of vesicles in the luminal periphery. Spermatophores were absent in the sections of minor coil in the specimens studied. In immature specimens,


Fig. 11—Luminal periphery of the median vas deferens (MVD) under active secretion. Small and large electron-dense bodies (eb & lb), microvilli (Mv), vesicles (arrowheads) and rough endoplasmic reticulum (rer) are visible. The lumen (LU) with homogeneous secretion (hs) is also seen. Scale bar = 2 lm.

there was no differentiation into major or minor coils, and the lumen was devoid of sperm cells. Posterior vas deferens. The PVD possessed the same diameter as that of the minor coil of the MVD at the beginning, but tapered towards the end. The layers of the lumen wall remained the same as that of the MVD. Here, also, the columnar cell lining had numerous foldings forming pouches (Fig. 13). The number of pouches increased so much that the adjacent ones pushed each other at their ‘mouth’ forming a void. The columnar cells continued to multiply, pushing the basophilic spindleshaped nuclei further to the base, thus accommodating more cells. The lumen was filled with granular frothy eosinophilic matrix, and sperm cells were absent.

Fig. 10—Major coil of median vas deferens (MVD) showing spermatophores (Sph) bathed in spermatophoric matrix (spm). Columnar epithelial cell lining (Co) and pouch-like folds (F) of the epithelial lining are also visible. Scale bar = 100 lm.

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Ejaculatory duct and penis. The ejaculatory duct was circular in outline, with a central lumen lined by epithelial layer. The outermost layer was made up of muscle tissue, which was covered by connective tissue (Fig. 14). The lumen was filled with eosinophilic matrix and was devoid of sperm cells. The ejaculatory duct was followed by a flexible, muscular penis covered by a thin layer of cuticle (Fig. 15). © 2012 The Authors Acta Zoologica © 2012 The Royal Swedish Academy of Sciences



Fig. 14—Ejaculatory duct showing lumen (LU), columnar cell lining (Co) of the lumen and external muscle layer (M). Scale bar = 100 lm.

Fig. 12—Lobated nucleus (N) with electron-dense nucleoli (arrowheads) in the columnar cells of the minor coil of median vas deferens (MVD). Arrays of rough endoplasmic reticula (rer) are also seen. Scale bar = 4 lm.

Fig. 15—Penis showing lumen (LU), muscle layer (M) and outermost cuticle layer (Cut). Scale bar = 200 lm.

Discussion

Fig. 13—Posterior vas deferens (PVD) showing increased number of epithelial pouches (P) and lumen containing granular eosinophilic matrix (M). Columnar cells (Co) lining the lumen and their nuclei (arrowheads) are also seen. Scale bar = 50 lm. © 2012 The Authors Acta Zoologica © 2012 The Royal Swedish Academy of Sciences

The structure of the male reproductive system of P. pelagicus closely agrees with the anatomical descriptions of various brachyuran crabs like P. sanguinolentus (Ryan 1967), C. sapidus (Johnson 1980), C. smithii (Balasubramanian and Suseelan 2000), G. cruentata (Tatiane and Silva 2006), U. cordatus (Castilho et al. 2007) and B. cunicularis (Sherkhane et al. 2010). The testicular lobules were delimited by a thin membraneous layer, which is supposed to be squamous epithelium as in G. cruentata (Tatiane and Silva 2006) and U. cordatus (Castilho et al. 2007). The lobules were called as ‘testicular cysts’ in G. cruentata (Tatiane and Silva 2006). The doublelayered wall of testis made of an outer connective tissue and inner delicate epithelial layer in the present study matches with the observations in P. sanguinolentus (Ryan 1967) and C. smithii (Balasubramanian and Suseelan 2000).

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The seminiferous tubule at the testis region was roughly oval in shape and was lined with columnar cells in the present study. This is concurrent with the observations in P. sanguinolentus (Ryan 1967), C. smithii (Balasubramanian and Suseelan 2000) and C. borealis (Moriyasu et al. 2002). The commissure connecting the two limbs of the testis as observed in many crabs by various authors is assumed to transfer sperms between the two limbs of the testis (Ryan 1967). The size of the sperm cells decreased with the progress of spermatogenesis initially in the present study. The spermatozoids of U. cordatus were only about 36% the size of the primary spermatogonium, and this was attributed to the reduction in the size of cytoplasm due to the condensation of chromatin (Castilho et al. 2007). The synchronous type of sperm development with sperm cells in the adjacent lobules under closely similar developmental stages, as observed in P. pelagicus, agrees to the observations in U. cordatus (Castilho et al. 2007) and B. cunicularis (Sherkhane et al. 2010). Although the lobules were found to have cells in the same stage, the lobes had lobules under different stages of development. Therefore, it is reasonable to assume from the pattern of spermatogenesis that there may be some periodicity for the formation of spermatogonia, with a specific duration for it to become a mature sperm. Even though the seminiferous tubule is accessible to all lobules in a lobe, only lobules with spermatozoa seem to evacuate the contents into the lumen. This assumption is supported by the report in U. cordatus where only the mature spermatozoids are found within the seminiferous tubules (Castilho et al. 2007). The vas deferens was divided into three parts in the present study. This is in agreement with the classifications in P. sanguinolentus (Ryan 1967), L. emarginata (Hinsch and Walker 1974), C. sapidus (Johnson 1980), C. smithii (Balasubramanian and Suseelan 2000) and U. cordatus (Castilho et al. 2007). A three-layered wall for AVD as in the present study was reported also in P. sanguinolentus (Ryan 1967) and C. smithii (Balasubramanian and Suseelan 2000). The lumen of the vas deferens was lined by a layer of glandular epithelium and towards the exterior was a thick layer of muscle sheath in U. cordatus (Castilho et al. 2007) and B. cunicularis (Sherkhane et al. 2010). In the AVD of P. pelagicus under the present study, the difference in the cells lining the inner epithelium in the proximal and distal portions may be for producing different types of amorphous substances that enveloped the spermatophores as reported in S. serrata (Uma and Subramoniam 1984). A similar change in the epithelial lining of AVD from cuboidal to columnar was reported in C. borealis (Moriyasu et al. 2002), the penaeiodean shrimp Sicyonia ingentis (Subramoniam 1995) and the red claw crayfish Cherax quadricarinatus (Lopez-Greco et al. 2007). Folds with tall columnar cells seen at the proximal part of AVD in the present study were supposed to increase the surface area of the active secretory epithelial lining (Ryan 1967). Sperm cell aggregations noticed in the distal portion of AVD as also reported in P. sanguinolentus (Ryan 1967) are the precursors of spermatophores bathed

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in spermatophoric matrix. Lobated nuclei observed in the columnar epithelial cells in present study denote stress on the nucleus due to high activity as suggested in S. ingentis (Subramoniam 1995). The high incidence of various cell organelles and the presence of secretory vesicles and moderately electron-dense spheres at the luminal periphery evidenced high exocytotic activity as reported in Cherax sp. (Talbot and Beach 1989), the grey shrimp Litopenaeus setiferus (Ro et al. 1990) and S. ingentis (Subramoniam 1995). The smaller vesicles might have fused to form high-density secretory granules. The presence of blebs, formed by the cell membrane at the luminal periphery in the present study, evidences the transfer of materials across the columnar cell membrane by the process of blebbing as explained in U. cordatus (Leite 2002). In E. asiatica, the cell membrane of the glandular epithelial cells ruptured to release the cytoplasmic contents into the lumen (Subramoniam 1984). The large confluence of microvilli projecting into the lumen from the epithelial lining of the AVD seen here is similar to the reports in the hermit crab Micropagurus acantholepis in which the microvilli facilitated the transfer of materials between lumen and epithelial cells (Tudge and Lemaitre 2004). Most of the anterior part of the vas deferens was involved in the secretion of substances required to envelope and store spermatophores in the Spanish lobster S. chacei (Hinsch and McKnight 1988). The histological sections showed two matrices with different staining properties, surrounding the spermatophore in the lumen as observed in P. sanguinolentus (Ryan 1967). The heterogeneous matrix consisted of basophilic vesicles and granules secreted by the epithelial columnar cells, and the smooth homogeneous layer filling the lumen was produced by the coalescence and condensation of the former (Subramoniam 1995). Spermatophore formation and fluid secretion were concomitant at the AVD in S. chacei (Hinsch and McKnight 1988). The glandular epithelial cells lining the lumen of AVD secrete a kind of fluid that serves in the making of spermatophores and as a medium for transporting spermatophores in B. cunicularis (Sherkhane et al. 2010). The microvilli projections in the MVD here closely resemble the descriptions in S. ingentis (Subramoniam 1995) and C. smithii (Balasubramanian and Suseelan 2000). In S. ingentis, the microvilli at MVD were relatively smaller than those in AVD probably due to its limited role as an absorptive organ alone (Subramoniam 1995). However, in the present study, the microvilli were envisaged to participate in the secretory activity also as indicated by an increased incidence of cell organelles. An opaque, adhesive, mucoid material, termed as seminal secretion, found to be extruded from the MVD of Clibanarius vittatus (Hess and Bauer 2002) and U. cordatus (Castilho et al. 2007), acted as a medium for storing spermatophores. A similar material was noticed in the present work as well. The increased musculature and number of pouches observed in the minor coil of P. pelagicus here indicated high secretory and a possible absorptive activity as in C. smithii (Balasubramanian and Suseelan 2000). © 2012 The Authors Acta Zoologica © 2012 The Royal Swedish Academy of Sciences


The increased number of folds on the lumen wall and high prevalence of vesicles at the luminal periphery of the PVD indicated the secretory nature of this region as observed in U. cordatus (Castilho 2007). In S. ingentis, the lumen of the PVD was narrowed due to the increase in thickness of the epithelial lining of vas deferens and the encircling musculature (Subramoniam 1995). The peripheral pouches of the PVD are thought to play a role in the formation of the gelatinous material that baths the mature spermatophores as disclosed by the basophilic vesicular layer adjoining the columnar cells in the present study. In the PVD here, the sperm masses were not noticed in the lumen because they were dispersed by the dilution effect of the fluid secreted, as explained in S. ingentis (Subramoniam 1995). The frothy granular matrix in the PVD observed in the present study is supposed to have been secreted by the epithelial layer as reported in A. symnista and E. asiatica (Subramoniam 1984). The precise sites of the production of the two different matrices observed here are not explained fully so far. However, two types of matrices have been reported in the same species by El-Sherief (1991), who studied the structure of spermatozoa and spermatophore of P. pelagicus. Simple columnar epithelium lining the seminiferous tubule of the testis has been suggested as the site in G. cruentata (Garcia and Silva 2006). The structure of the ejaculatory duct with a roughly circular outline observed in P. pelagicus is in accordance with the descriptions in other brachyuran crabs (e.g. Ryan 1967; Subramoniam 1984, 1995; Balasubramanian and Suseelan 2000; Castilho et al. 2007; Sherkhane et al. 2010). In the hermit crab Dardanus asper, the spermatozoa formed a continuous stream as they left the testicular region and were mixed with epithelial secretions in the vas deferens. During this mixing process, the spermatozoa were grouped and separated into distinct and visible spermatophores within the coiled vas deferens by means of contractions of the surrounding muscular layer. The contractions moulded the seminal secretions surrounding the grouped spermatozoa into the characteristic shape of the spermatophore (Mathews 1953). A similar process is envisaged in P. pelagicus in the present study. The present study on P. pelagicus helped to understand the structure of various parts of the male reproductive system by providing both light microscopic and ultrastructural details. The study also indicated the active participation of the various regions of vas deferens in the formation of spermatophores through electron micrographs. A clear understanding of the structural details, cyclic changes, process of sexual maturation and the sequence of spermatogenesis is envisaged to help in framing out and standardizing the methodologies for broodstock management and seed production of this commercially important species. Acknowledgements The authors wish to express their sincere thanks to Dr. G. Syda Rao, Director, Central Marine Fisheries Research Insti© 2012 The Authors Acta Zoologica © 2012 The Royal Swedish Academy of Sciences


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© 2012 The Authors Acta Zoologica © 2012 The Royal Swedish Academy of Sciences