Ultrastructure Developments During Spermiogenesis in Polydora ciliata (Annelida: Spionidae), a Parasite of Mollusca

GAO Yan, ZHANG Tao ZHANG Libin QIU Tianlong, XUE Dongxiu, and YANG Hongsheng

1) Key Laboratory of Marine Ecology and Environmental Sciences, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, P. R. China

2) University of Chinese Academy of Sciences, Beijing 100049, P. R. China

Ultrastructure Developments During Spermiogenesis in Polydora ciliata (Annelida: Spionidae), a Parasite of Mollusca

GAO Yan1),2), ZHANG Tao1), ZHANG Libin1), QIU Tianlong1),2), XUE Dongxiu1),2), and YANG Hongsheng1),*

1) Key Laboratory of Marine Ecology and Environmental Sciences, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, P. R. China

2) University of Chinese Academy of Sciences, Beijing 100049, P. R. China

Spionid worms of Polydora ciliata inhabit the shells of many commercially important bivalves and cause disease in molluscan aquaculture. Their sperm structure is closely related to their fertilization method. To give an insight into the sperm structure and spermatogenesis, ultrastructure details of the subcellular components of germ cells during spermiogenesis of Polydora ciliata are detected by transmission electron microscopy (TEM). In P. ciliata, during spermiogenesis, chromatin is regularly arranged as dense fibrils and becomes more condensed when the nucleus elongates. Microtubules do not surround the nucleus during its elongation. The Golgi phase is characterized by the formation of proacrosomal granules within the Golgi apparatus. The proacrosomal granules fuse to form a single, spherical acrosomal vesicle that migrates to the anterior pole of the cell. At the time of nuclear condensation, mitochondria become reduced in number but increased in size, causing deep indentation at the base of the nucleus. The mid-piece has a few mitochondria. The cap phase includes the spreading of the acrosomal granule over the surface of the nucleus of the differentiating spermatid. The acrosomal phase of spermiogenesis is typically associated with changes in the shape of the nucleus, acrosome and tail. The relationship of sperm ultrastructure to spermiogenesis in spionidae species was discussed.

Polydora ciliata; spermiogenesis; ultrastructure

1 Introduction

The Polydora complex in the family of Spionidae contains at least 115 species in nine genera: Amphipolydora, Boccardia, Boccardiella, Carazziella, Dipolydora, Polydora, Polydorella, Pseudopolydora, and Tripolydora (Rice et al., 2008). These genera, commonly known as blisterworms or mudworms, share a setigerous fifth chaetiger which is modified and includes specialized setae or spines. These spines are useful in tube construction and maintenance. These worms can be troublesome parasites of commercially important bivalves. The boring behavior in the shells induces the creation of blisters by shell marterial secretion (conchiolin covered with nacre) to isolate the worm. These blisters containing the compact sludge can occupy a large proportion of the mantle cavity space and contribute to weakening the shell. Worm infestation affects the presentation of half-shell and reduces the market value.

Several species of polydorid have been reported to cause damage to abalone (Lleonart et al., 2003; Mcdiarmid et al., 2004; Simon et al., 2004; Aviles et al., 2007), oysters (Loosanoff and Engle, 1943; Mohammad, 1972; Sato-Okoshi and Okoshi, 1993; Nel et al., 1996), scallops (Sato-Okoshi et al., 1990; Bower et al., 1992; Sato-Okoshi and Okoshi, 1993; Mortensen et al., 2000; Silina, 2006), clams (Riascos et al., 2008) and many other mollusks. Infestation by Polydora, therefore, is an important problem in molluscan aquaculture.

About two decades ago the Japanese scallop Patinopecten yessoensis was introduced from Japan to China, and became one of the most important commercial bivalve mollusks (Li et al., 2007). However, numerous Polydora blisters were found in the scallop culture (Read et al., 2010; Walker et al., 2011; Sato-Okoshi et al., 2012; Silina et al., 2012; Souto et al., 2012). A scallop devotes much of its energy in secreting nacre and concholin during mud blister formation, which leads to poor quality and low growth rate. Clarifying the life cycle, including reproductive biology, of these boring Polydora could help in developing a management strategy to minimize the impact of mud worm infestation of scallop culture. Thereproductive biology of Polydora is poorly understood because of their life history characteristics. For example, tube-dwelling lifestyles limit direct body-contact or decrease the mobility of individuals so that encounters between sexes are infrequent or impossible. Due to the relationship between structural features of the spermatozoa and biology of fertilization of a species, the study of the sperm ultrastructure of Polydora could elucidate the mode of sperm transfer in this species.

The ultrastructure of spermatogenesis has been reported in three species of Polydora only: Polydora ligni, P. websteri, and P. socialis (Rice, 1981). Similar characteristics such as a conical acrosome with a distinctive substructure, and the presence of platelet-shaped electron-dense bodies throughout the nuclear region and mid-piece, have been described. Nevertheless, the structure of the spermatozoa of individual species has unique features. The present work describes the ultrastructure of spermiogenesis and sperm in Polydora ciliata (Johnston, 1838), a parasite of the scallop Patinopecten yessoensis (Bivalvia, Mollusca) in the Yellow Sea.

2 Materials and Methods

Scallops infested with P. ciliata were collected from the Yellow Sea (39˚03´N, 122˚44´E) in March 2008. Adult worms, removed from the shell fragments with forceps, were relaxed in isotonic magnesium chloride to examine the gametogenic segments. Middle gametogenic segments were cut off and fixed in cold (4℃) 2.5% glutaraldehyde in 0.1 mol L-1sodium cacodylate buffer (pH 7.4) containing 3% sucrose, and left overnight at 4℃. Then the tissues were postfixed in 1% osmium tetroxide for 1 h, rinsed in 0.1 mol L-1sodium cacodylate buffer at pH 7.4, dehydrated in an ascending series of ethanol, and infiltrated and embedded in Epon 812. Ultrathin sections (55–70 nm) at different levels were cut on an LKB ultramicrotome, mounted on bare 200-mesh copper grids, and double-stained with uranyl acetate (5 min) and lead citrate (3 min); all staining was carried out in a 60℃ oven. The specimens were then examined in a transmission electron microscope (Hitachi, H-700) operating at an accelerating voltage of 100 kV.

3 Results

Different developmental stages of gametes, from spermatocytes to mature spermatozoa, were found in the seminal vesicles of each investigated mature individual. We divide spermiogenesis into three stages and describe them separately for convenience.

3.1 Early Stage

The presence of synaptonemal complexes is an important characteristic of primary spermatocytes. It indicates that the cells are in the leptotene stage of the first meiotic division. The spherical nuclei of primary spermatocytes occupy almost the entire cell volume.

Fig.1 TEM micrographs showing early stages of spermatogenesis in P. ciliata. A, A primary spermatocyte showing a large nucleus with several synaptonemal complexes (arrowhead); B, A secondary spermatocyte with numerous mitochondria; C, A secondary spermatocyte with diffuse heterochromatin in the nucleus. m, mitochondria; N, nucleus; NE, nuclear envelope; pg, proacrosomal granule. Scale bars = 1 μm.

Numerous dense granules and small mitochondria are scattered in the cytoplasm (Fig.1A). The secondary spermatocytes are similar to the primary spermatocytes in appearance, and their cytoplasm contains numerous mitochondria. The nuclei of the secondary spermatocytes appear to be slightly smaller and contain more heterochromatin than those of the primary spermatocytes. Vacuoles are present in the cytoplasm, and the ovoid mitochondria are close to the nucleus (Fig.1B). Coarsely granular chromatin begins gathering into a cluster. Nuclear pores are abundant. Two electron-dense granules which are recognized as proacrosomal granule, appear in the secondary spermatocytes (Fig.1C). The proacrosomal granules are large and ovate, and migrate towards the opposite pole of the cell.

3.2 Middle Stage

A secondary spermatocyte forms two spermatids after the second meiotic division. The cellular components change synchronously during the differentiation of the spermatids. We describe these changes separately for convenience.

Fig.2 TEM micrographs showing mid- to late spermatids in P. ciliate. A, A mid spermatid with nucleus (n) and developing axoneme (ax); B, A mid spermatid with Golgi complex (g) and acrosome-like structure (ac); C, A mid spermatid with trapeziform nucleus; D, A mid spermatid with a centriole lying close to the plasmalemma; E, Late spermatid with proximal centriole; F, Late spermatid with distal centriole. ac, acrosome-like structure; ax, axoneme; c, centriole; dc, distal centriole; N, nucleus; m, mitochondria; pc, proximal centriole; pg, proacrosomal granule; pl, platelet-shaped electron-dense body. Scale bars = 1 μm.

Nuclear Condensation During this stage of spermiogenesis, the condensation of chromatin proceeds and the nucleus then becomes trapeziform (Fig.2A). The granular chromatin condenses into tightly packed and uniformly electron-dense strands. These dense strands are in a progressive manner from anterior to posterior in the nucleus (Fig.2B). The chromatin then becomes regularly arranged as dense fibrils (Fig.2C). A centriolar fossa forms at the posterior end of the nucleus. The fossa becomes progressively deeper as nuclear elongation proceeds (Fig.2E).

Proacrosome Formation In the early spermatids, the proacrosomal granules come to lie close to the developing anterior of the nucleus in a slight nuclear depression (Fig.2A). Acrosome formation is augmented with the appearance of a spherical, membrane-bound pro-acrosome granule on the concave surface of the Golgi complex between the Golgi and the nucleus. The location of the Golgi region and pro-acrosomal granule defines the future anterior end of the sperm cell. This granule subsequently fuses with the cell nucleus and generally contains a region of denser material (Fig.2A). With the differentiation of the spermatids, the proacrosomal granules fuse to form a single, spherical acrosomal vesicle that migrates to the anterior pole of the cell. This vesicle elongates and finally locates in the cup-shaped anterior surface of nucleus. At the end of the acrosome phase, the proacrosomal granules merge and become a long, narrow structure that forms a kind of cup beneath the plasma membrane (Fig.2B).

Cytoplasmic Inclusion In early spermatids, a few mitochondria and multivesicular bodies are distributed at the margins of the cytoplasm. At the time of nuclear condensation, mitochondria become reduced in number but increase in size; even when they become apposed to the nuclear surface, they still undergo an increase in size, causing deep indentations at the base of the nucleus (Figs.2C, E). The Golgi complex is found in the posterior cytoplasm of early spermatids, opposite to the primary acrosomal vesicle (Fig.2B). Electron-dense platelet-shaped bodies form a layer around the developing spermatid just beneath the plasma membrane (Fig.2D).

Axoneme Formation The formation of the centriole occurs simultaneously with the condensation of chromatin in the nucleus. In early spermatids, the centrioles lie close to the plasmalemma and lack a prominent centriolar satellite (Fig.2D). During chromatin condensation, the flagellum develops from the centrioles. The axoneme becomes longer and bent because of continuous growth. As a result, an axoneme is usually sectioned twice within one cell (Fig.2F). The distal centriole and proximal centriole are oriented at right angles to each other in late spermatid. The fossa at the posterior end of the nucleus is invaded by the proximal and distal centrioles that fuse to form the basal body of the flagellum. The proximal centriole lies under the posterior fossa of the nucleus while the distal centriole is associated with the axonemal complex with microtubules connecting with the flagellum (Figs.2E, F).

3.3 Late Stage

Late spermatids can be recognized by a more elongate nucleus, while the chromatin is becoming homogeneously electron-dense. The nucleus has a posterior fossa that houses the proximal centriole. The centrioles are located at the base of the nucleus in a position opposite to the acrosome. The acrosome has reached its final position and shape, and the mid-piece mitochondria are located around the axoneme. The distal part of the axoneme, which has a 9 + 2 arrangement of microtubules, is located centrally within the flagellum with only a small amount of surrounding cytoplasm (Fig.3H).

Early in spermiogenesis, the proacrosome lies next to the plasmalemma, separating from the nucleus (Fig.3A). Later, the proacrosome migrates to the surface of the nucleus so that the prominent electron-dense zone becomes apposed to the nuclear envelope (Fig.2B). The proacrosome then becomes cup-shaped, with a well-defined acrosomal fossa. Longitudinal microtubules appear in the developing acrosomal vesicle (Fig.3A).

At the time of acrosome formation, the mitochondria become reduced in number but increased in size. The internal membranes in the sausage-shaped mitochondria are clearly discerned during earlier stages (Fig.3B). Initially, most mitochondria are almost circular in crosssection. The mid-piece contains up to seven mitochondria, six being circular and one being bean-shaped. The mitochondria lie close together and the zone contacting with the membranes are rather electron-dense (Fig.3G).

Amoeboid-shaped cells with a large nucleus are found during spermiogenesis. They are called auxiliary cells. The cytoplasm contains a Golgi complex, a few cisternae of endoplasmic reticulum and mitochondria. These cells may support nutrients to the sperm (Fig.3I).

4 Discussion

4.1 Spermiogenesis Characteristics

The present results revealed substantial similarities in the ultrastructure of the spermiogenesis and spermatozoa among P. ciliata from the Yellow Sea and the three species from the Indian River Lagoon in the United States (Rice, 1981). In Polydora, the microtubules are absent during the granular chromatin condensation into tightly packed and uniformly electron-dense clumps. Chromatin structure of the same features was found in Boccardiella hamata from the Sea of Japan (Reunov et al., 2009).

In most taxa, the acrosome is a cap-like structure derived from the Golgi complex (Zhu et al., 2008). However, it is different for P. ciliata. In this species, the Golgi complex forms the proacrosomal granules, which fuse to form an acrosomal vesicle. This structure is similar to that found in Syllis krohni (Musco et al., 2008).

In addition to the elongated sperm head in Polydora, the presence of cytoplasmic platelets may be important in the biology of reproduction. If these platelets are energy storage organelles (as hypothesized), then reproduction in this species could include prolonged storage of sperm without loss of viability, multiple egg laying following a single sperm transfer, establishment of new populations by a single inseminated female, and the elimination ofsimultaneous spawning of males and females. These reproductive adaptations could be quite advantageous to opportunistic species such as P. ligni and S. benedicti, which have rapid reproduction and tend to colonize ephemeral habitats (Grassle, 1974).

4.2 Correlating Sperm Morphology and Reproductive Mechanisms

Wilson (1991; see also Giangrande, 1997) reviewed sexual reproduction in the Polychaeta and identified 17 modes based on the type of larvae and the mode of development. He categorized the species as using free spawning (external fertilization) with no care of larvae, brooding of larvae (four types), or using gelatinous encapsulation of larvae. Offsprings were released as lecithotrophic larvae, planktotrophic larvae, or larvae that underwent ‘direct development’. This variability in reproductive mode is reflected in the variety in sperm structurethat is found among polychaetes. Ultrastructure studies of spermiogenesis and the morphology of spermatozoa in polychaetes have been reviewed (Eckelbarger and Grassle, 1987; Wilson, 1991; Giangrande, 1997; Rouse, 1999). A number of studies on ultrastructure aspects of spermatozoa in polychaetes have shown a large diversity in sperm morphology (Tzetlin et al., 2002; Jouin-Toulmond and Purschke, 2004; Simon and Rouse, 2005; Lepore et al., 2006; Musco et al., 2008; Reunov et al., 2009). The hypothesis that sperm structure correlates with the method of sperm transfer or biology of fertilization is supported by these studies. Sperm transfer in polychaetes occurs in two main modes: non-aggregate transfer, in which sperm swim freely and are not packed together before reaching eggs; and aggregate transfer, in which sperm are packed together by varying complex structure before reaching eggs. Life history characteristics and habitat choice have been considered strong selective forces for the mode of sperm transfer. For example, sessile or tube-dwelling lifestyles limit direct body-contact or decrease the mobility of individuals, so that encountering between different sexes are infrequent or impossible. Thus, neither copulation, nor pseudocopulation, nor indirect hypodermic impregnation would be favored. Broadcast spawning or free transfer of spermatophores may be the only alternative for such species. However, broadcast spawning requires a large number of gametes and synchronous reproduction in the population (Hsieh and Simon, 1990). In most cases, sperm that is directly introduced comprises a narrow bell-shaped acrosome, a elongated nucleus with highly condensed chromatin, a typical 9+2 flagellum derived from the distal centriole, and a few mitochondria as a sheath around the axoneme (Jamieson and Rouse, 1989; Purschke and Fursman, 2005).

From the results in this study, the sperm structure of P. ciliata resembles the direct sperm transfer type. The acrosome contains a compact vesicle and an axial rod (Figs.3A, 4A). The chromatin is completely condensed in the spermatozoa. The nucleus has a posterior fossa that houses a flagellum derived from proximal centriole (Fig.4A). The sperm mitochondria are spherical during spermiogenesis and elongate slightly down the axoneme, forming a tight sheath in very late spermatid (Fig.4D). Because the mitochondria may continue to fuse during the late spermatid stage, the number of mitochondria in middle-piece is not clear and more detailed study is required. The elongation of the nuclei and mitochondria may cause the sperm to be more effective (Rice et al., 1992).

The present study has shown that features such as the regularly arranged fibrous chromatin, the formation of proacrosomal granules, and the presence of cytoplasmic platelets are important features of P. clilata. Jamieson (1989) divided polychaete sperm into ect-aquasperm, ent-aquasperm, and introsperm. His report on the sperm structure had been demonstrated for the Spionidae. Our research supports his conclusion. Shell-boring spionid polychaetes are ubiquitous in many mollusca species and there are no effective treatments. Maybe we can explore some measures to prevent and control this parasite of Mollusca from fertilization.

Fig.4 Reconstruction of the spermatozoon of P. ciliata. A, longitudinal section; B, transverse section through the acrosome; C, transverse section through the nucleus; D, transverse section through the mid-piece; E, transverse section through the flagellum; ax, axoneme; m, mitochondria; n, nucleus. Scale bar = 1 μm

Acknowledgements

The work was supported by the National Key Technology Support Program (2011BAD13B05). We appreciate the technical help from Prof. Ming Jiang and Mrs. Jialin Xie.

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(Edited by Qiu Yantao)

(Received February 25, 2013; revised March 24, 2013; accepted August 29, 2014)

© Ocean University of China, Science Press and Spring-Verlag Berlin Heidelberg 2014

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E-mail: hshyang@qdio.ac.cn