Ultrastructural Investigations of Eusperm- atogenesis and Euspermatozoa in obtusa (Lamarck 1822) (: ) Jintamas Suwanjarat*1 & Waltraud Klepal2

1 Department of Biology, Prince of Songkla University, Hat-Yai, Thailand 90110. 2 Institute of Zoology, University of Vienna, Althanstr. 14, A-1090 Vienna, Austria.

*Author to whom correspondence should be addressed. E-mail: [email protected]

Abstract

Abstract. Species of the Potamididae occupy the full range of aquatic habitats and differ not only in the morphology and size of their shells but also in sperm morphology. In the past, several species were classified as . Characters of the developing and the mature spermatozoa have been used to gain better insight into their .

Cerithidea obtusa (Lamarck 1822) is the most dominant brackish water gastropod of the forests in Southern Thailand. Spermatological data are scarce in this species. In order to confirm its taxonomic position within the Potamididae, euspermatogenesis and euspermatozoa are examined by transmission electron microscopy. The morphological changes during spermiogenesis such as nucleus condensation, acrosome formation and development of the midpiece are described.

Problem

The Potamididae is a large and important family of which varies greatly in shell characters and shell size. Houbrick (1991)

commented that because of the unawareness of the significant anatomical differences among cerithioidean gastropods many Potamididae were erroneously classified as Cerithiidae. In addition, many genera of each potamidid subfamily were mistaken for each other and for Cerithiidae. The modern classifications of prosobranchs, to which the Caenogastropoda belong, are generally achieved by light- and electron microscopy (Haszprunar, 1988). In particular, studies of mature spermatozoa and spermiogenesis have provided insight into characters which have become incorporated into prosobranch systematics (Franzén, 1955, 1970; Healy, 1988a). The morphological diversity of spermatozoa in prosobranchs has been considered as a guide to judge the phylogenetic and taxonomic relationships within molluscs. Many authors have confirmed the taxonomic position of prosobranch gastropods by considering sperm-data (Morton & Young, 1964; Healy, 1983; Robertson, 1985; Healy, 1988b, c; Minniti, 1993). Although a number of recent investigations have dealt with sperm ultrastructure of the superfamily Cerithioidea, information on sperm- atogenesis of euspermatozoa of potamidid gastropods is scarce (Healy, 1982b, 1986a, b; Attiga & Al-Hajj, 1996), and a detailed description of the early stages of spermatogenesis at the ultrastructural level is missing. The present investigation therefore aims to fill this gap by providing a detailed description of spermatogenesis and the mature euspermatozoon of the potamidid Cerithidea obtusa (Lamarck 1822). Similarities and differences in the spermatozoa of C. obtusa will be correlated with those of closely - related species as well as with other caenogastropods.

Apart from providing further evidence for the phylogenetic relationship of this species, this study also has ecological implications. Cerithidea obtusa is one of the most dominant and conspicuous brackish water gastropods found on the mudflats in the mangrove forests of Thailand and is therefore used as an index of fertility. Despite their abundance, accessibility and economical importance as food (Amornjaruchit, 1988), their biology, and especially their reproductive biology, is hardly known. Attempts have been made to examine their ecological adaptation, population dynamics, feeding activity (Kasinathan & Natarajan, 1981; Kitting, 1989) and even radular morphology (Suwanjarat, 1994; Suwanjarat & Suwaluk, 1997 observing was carried out at the Institute of Zoology, University of Vienna, Austria.

In Cerithidea obtusa, as in many other caenogastropods, spermatogenesis occurs in the testes, in which the different developmental stages are found.

1. Spermatogonia

Spermatogonia are close to the wall of each germinal follicle. The large, round nucleus of the spermatogonium has one or two nucleoli. Small clumps of electron-dense chromatin are loosely distributed in the nucleoplasm (Fig. 1). The narrow rim of cytoplasm around the nucleus contains a few rounded mitochondria, a small amount of endoplasmic reticulum and a Golgi body.

2. Spermatocytes

The primary spermatocytes of Cerithidea obtusa are roughly spherical to irregular in shape. Their round or ovoid nuclei contain patchy chromatin with one large and highly electron-dense nucleolus. There are many pores in the nuclear membrane. The amount of cytoplasm around the nucleus is greater than in the previous stage; several round mitochondria and electron-dense inclusions are scattered within it. Rough endoplasmic reticulum, arranged concentrically, surrounds one large electron-dense granule (Fig. 2).

The secondary spermatocytes are smaller than the primary spermatocytes. Their nuclei are round with some electron-dense chromatin dispersed throughout the nucleoplasm and there are numerous pores in the nuclear membrane. The cytoplasm is granular and contains a well-developed Golgi body, round mitochondria, rough endoplasmic reticulum and some moderately sized electron-dense granules. The developing spermatocytes are connected by intercellular bridges (Fig. 3).

3. Spermatids

The spermatid may be distinguished from the spermatocyte by its general denser appearance and by the numerous organelles in the cytoplasm. During spermiogenesis the spermatids undergo differentiation to become mature spermatozoa. The following major steps are taken: nucleus condensation, acrosome formation, development of midpiece and axonemal complex. a. Nucleus condensation

The early spermatid has a spherical, eccentric nucleus. There are still many pores in the membrane. The nucleoplasm is granular with some aggregation of heterochromatin and thus it appears more electron- dense than in the previous stages (Fig. 4). The nucleoplasm progressively accumulates electron-dense chromatin, concomitantly with the change of the nuclear shape to irregular (Figs. 5, 6). As spermiogenesis proceeds, the granular chromatin thickens at the periphery and begins to accumulate at the posterior nuclear pole. The nucleus is compressed posteriorly and laterally expanded; it has its greatest diameter perpendicular to the flagellar axis. The nucleus is now in its fibrous phase with parallel fibrillar appearance of the chromatin (Fig. 7). The granular cytoplasm of the early spermatid shows a Golgi body surrounded by vesicles, several large mitochondria and rough endoplasmic reticulum. Cytoplasmic bridges connecting spermatids are often observed (Fig. 8 electron-dense fibrils which are elongated and oriented longitudinally along the nuclear vertical axis. At the basal pole of the nucleus is a deep invagination, the indendation fossa, with the axoneme insertion (Figs. 9, 13). As the next step of nuclear condensation the chromatin fibrils thicken and form lamellae (Figs. 10, 14). During further development the nucleus lengthens and the chromatin lamellae thicken, leaving only a few spaces between them (Fig. 11). The condensation is clearly correlated with nuclear elongation. At the late spermatid stage, the nucleus is columnar with a length to breadth ratio of no more than 4 : 1. b. Acrosome formation

Acrosome formation begins with the proacrosomal vesicle arising from the Golgi apparatus concomitantly with the nuclear condensation (Fig. 15). The proacrosomal vesicle elongates and differentiates to the pre-attachment acrosome. It is surrounded by microtubules oriented longitudinally; parallel to its wall it has an inner supporting structure and at one end of the vesicle there is an electron-dense granule (Fig. 16). The vesicle develops into the hollow acrosomal cone with the electron-dense granule fixed to its base (Fig. 17). This pre- attachment acrosome moves anteriorly towards a slight apical depression of the condensing nucleus (fibrous phase), then it tilts and moves into a vertical position (Fig. 18). As it begins to tilt, the large dense granule separates from the base of the acrosomal cone and attaches to the depression in the nuclear apex (Fig. 19). Here the dense granule flattens to form a basal plate with an acrosomal rod arising in its center. The internal supporting structure of the acrosomal cone disappears, while an external supporting structure appears and remains during the process of acrosomal development until it is gradually obscured in the mature acrosome. The acrosomal cone, which is invaginated at the base, is then placed upon the basal plate with its prominent acrosomal rod extending into the subacrosomal space (Fig. 20). The acrosome then undergoes pronounced elongation whereby the inner lining of the acrosomal cone thickens (Fig. 21). At the apex the tapering inner lining fuses, thus causing a constriction in the subacrosomal space. The external supporting structure still exists at this phase (Figs. 22, 23, 24). As development proceeds towards maturation the axial rod becomes increasingly obscured and scarcely appears in the mature acrosome. c. Development of midpiece and axonemal complex

In the early spermatid the mitochondria distributed throughout the cytoplasm begin to aggregate at the posterior pole of the nucleus. As the nuclear chromatin condenses they fuse to form four large, spherical mitochondria surrounding the axoneme (Fig. 8). In the course of the midpiece-development the mitochondria elongate and their cristae are oriented parallel. The axoneme is initially associated with a single, short, dense structure, presumably a centriole, which inserts into the indentation fossa at the base of the nucleus. The flagellum lengthens at the same time as the antero-posterior axis of the lamellar nucleus. The cytoplasm of this stage contains a well- developed Golgi body, rough endoplasmic reticulum, numerous vesicles and is rich in glycogen granules (Fig. 12 portion of the axoneme arises within the short posterior invagination of the nucleus. The mature acrosome consists of a long tapering cone resting on a thin basal plate capping the nuclear apex. An electron- dense, plate-like structure is noticeable at the beginning of the midpiece just behind the nucleus (Fig. 25). The midpiece of the euspermatozoon reveals four straight mitochondria of equal size around the axoneme. Each mitochondrial element is constructed of multiple, curved, parallel cristal plates. The proximal part of the tail shows the 9 + 2 pattern of microtubular arrangement surrounded by a sheath of glycogen granules inside the plasma membrane (Fig. 26). There is a circular dense band around the axoneme separating the midpiece from the glycogen piece of the tail (Fig. 27). The distal region of the tail lacks glycogen.

The euspermatozoon of Cerithidea obtusa (Potamididae) agrees well with the euspermatozoon of other Potamididae in the dimensions of the head and the midpiece. The present study shows that the early stages of spermatogenesis in Cerithidea obtusa euspermatozoa follow the general developmental pattern of modified euspermatozoa of caenogastropods as described by Maxwell (1983) and Voltzow (1994). The presence of only one moderately sized nucleolus in the nucleus of the primary spermatocyte in C. obtusa is so far unique for this species. The euspermiogenesis as seen in C. obtusa shows many features also common in other cerithioideans (Healy & Jamieson, 1981; Healy, 1982a, 1986b; Afzelius & Dallai, 1984; Afzelius et al., 1989; Hodgson & Heller, 1990; Minniti, 1993; Al-Hajj & Attiga, 1995; Attiga & Al-Hajj, 1996). However, the prominent rough endoplasmic reticulum seen in C. obtusa has never been described by other authors. Indeed, rough endoplasmic reticulum arranged in whorls is presumably, together with the small vesicles in the vicinity of the Golgi complex, responsible for the production of material for the formation of the acrosome. In some species of pulmonates the proliferation of the Golgi body and the amount of smooth endoplasmic reticulum during spermiogenesis have been suggested to play an important role in the process of acrosomal vesicle formation (Hodgson & Bernard, 1988; Voltzow, 1994).

The pattern of nuclear condensation during euspermiogenesis in C. obtusa– passing through a granular, fibrillar and lamellar phase as described by Henley (1973) and Maxwell (1983)– is similar to that demonstrated for other caenogastropods (e.g.,Healy, 1982b; Claveria & Etges, 1988). The mature nucleus of C. obtusa is rod-shaped and has a short basal invagination. The process of its formation closely resembles that studied in many other cerithioideans (Healy, 1982a; Afzelius & Dallai, 1983; Al-Hajj & Attiga, 1995; Attiga & Al-Hajj, 1996).

The acrosomal ultrastructure of gastropods may be species-specific and could thus provide useful taxonomic data as suggested by many investigators. The acrosome in the caenogastropods consists of three components: acrosomal cone, acrosomal axial rod and basal plate. These structures differ according to the shape and size of the acrosome. The acrosomal cone of the mature euspermatozoon of Potamididae is elongate and it varies in shape from truly conical to flat. The microtubules seen within the early stages of the developing acrosomal cone, and still existing in the pre-mature acrosome of C. obtusa, contrast with those of other potamidid snails, in which the microtubules are not associated with the developing acrosome (Healy, 1982b). But microtubules are present in the Cerithiidae bifasciata and C. tuberculatus (Attiga & Al-Hajj, 1996) and in some other caenogastropods (Buckland-Nicks & Chia, 1976

arranged around the acrosomal cone are thought to cause some rigidity and aid in the elongation of the cone, as was suggested in some non-cerithioidean caenogastropods (Walker & MacGregor, 1968; Buckland-Nicks & Chia, 1976). During acrosome formation in Cerithidea obtusa a dense internal supporting structure is found within the acrosomal cone, as reported in other cerithioideans like the potamidid ebeninus and the cerithiid species and C. tuberculatus (Healy, 1982a; Attiga & Al-Hajj, 1996). This structure is detected neither in the mature acrosome of Cerithidea obtusa nor in that of (Healy, 1982a) but it still exists in that of Clypeomorus bifasciata and C. tuberculatus. In the Thiaridae no internal supporting structure is seen within the acrosome.

The axial rod may consist of a single rod, multiple rods or diffuse deposits (Healy, 1982a, b and 1983). In Cerithidea obtusa the material of the single acrosomal rod is seen during the formation of the acrosome but is not present in the mature acrosome. In telescopium (Potamididae) some axial rod material is assumed to be present (Healy, 1983). Within the superfamily Cerithioidea, in the potamidid species Cerithidea obtusa the acrosome shape of mature euspermatozoa is antero-laterally compressed and similar to that of Clypeomorus bifasciata and C. tuberculatus (Cerithiidae; Attiga & Al-Hajj, 1996); it differs from the campanilid species of the superfamily Campaniloidea Campanile symbolicum (Healy, 1986b).

In acrosome formation and shape, Cerithidea obtusa is similar to some species of the Potamididae and Cerithiidae, whilst its midpiece more closely resembles most of the Potamididae, the Pleuroceridae, Scaliolidae and Thiaridae. In Cerithidea obtusa and all the families mentioned above, the midpiece of the euspermatozoon contains four straight mitochondrial elements with parallel cristal plates. The mitochondria are of equal size. Other potamidid species, e.g., Velacumantus australis (Healy, 1983) and Pyrazus ebeninus (Healy, 1982a), have two large and two small mitochondria, as found in the Cerithiidae, and Planaxidae – although in the latter the size difference is not pronounced (Healy, 1983; Hodgson & Heller, 1990).

The results of our investigation support Al-Hajj & Attiga (1995), who propose that the ultrastructure of euspermatozoa is of taxonomic significance at and above the family level, with evidence of some species-specific characters in certain cases. Apart from only one nucleolus in the nucleus of the primary spermatocyte and the presence of prominent rough endoplasmic reticulum throughout spermiogenesis, which have never been described before, the species- specific ultrastructural characters of Cerithidea obtusa are (1) an external supporting structure in the pre-mature acrosome, (2) microtubules in the early stages of the developing acrosomal cone and (3) the lack of an acrosomal rod in the mature acrosome.

Summary

The mature euspermatozoon of Cerithidea obtusa (Potamididae) has a long, tapering, antero-laterally compressed acrosomal cone; its nucleus is rod-shaped and has a short basal invagination as in most Cerithioidea. The midpiece has four equally sized straight mitochondria. The acrosome and its formation resemble that as described in some other Potamididae and Cerithiidae. A dense internal supporting structure present in C. obtusa during the formation of the acrosome only, still persists in the mature acrosome of the Cerithiidae. The midpiece is similar to that of most species of the Potamididae as well as the Pleuroceridae, Scaliolidae and Thiaridae. Species-specific characters of C. obtusa are microtubules surrounding the acrosome during its differentiation, an external supporting structure in the pre-mature acrosome and the lack of an acrosomal rod in the mature acrosome. The presence of only one nucleolus in the nucleus of the primary spermatocyte and the prominent rough endoplasmic reticulum found throughout spermiogenesis are notable.

Acknowledgements

The authors thank the Memorandum of Understanding (MOU) Programme, the ASEA-UNINET, the University of Vienna (Austria) and the Faculty of Science, Prince of Songkla University (Thailand) for financial support. Dr. Matthias Glaubrecht advised on the systematics of the Cerithioidea. We also thank Prof. Dr. Gerhard Haszprunar, Prof. Dr. Luitfried Salvini-Plawen and Dr. Gerhard Steiner for critically reading the manuscript. Dr. Marieluise Weidinger and Mag. Daniela Gruber are thanked for technical assistance in the Electron Microscopy Laboratory of the Institute of Zoology, University of Vienna.

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