ORIGINAL ARTICLE

Euspermatozoon structure and euspermiogenesis in cingulata (Gmelin, 1791) ( : )

Jintamas Suwanjarat1 and Sakol Suwaluk2

Abstract Suwanjarat, J.1 and Suwaluk, S.2 Euspermatozoon structure and euspermiogenesis in Cerithidea cingulata (Gmelin, 1791) (Caenogastropoda : Potamididae) Songklanakarin J. Sci. Technol., 2003, 25(4) : 413-422

The ultrastructural characteristics of euspermatogenesis and mature euspermatozoa of Cerithidea cingulata are described. Euspermatogonia possesses a large round concentric nucleus with one or two nucleoli. The spermatocyte is characterized by the abundant cytoplasmic organelles and eccentric nucleus with chro- matin distributed throughout as small granules. In the early spermatid, euspermiogenesis begins with the condensation of nucleus, the granular nuclear chromatin changes to fibrillar, lamellar and finally a homo- genous and highly electron dense nucleus. The cytoplasm of the early spermatid contains a well developed Golgi complex with many vesicles and a prominent rough endoplasmic reticulum located between the con- nection of the two daughter cells. Acrosome formation starts with proacrosomal vesicle which usually appears close to the well developed Golgi complex. This proacrosomal vesicle differentiates into a pre-attachment acrosome which then moves anteriorly towards the nucleus and finally attaches to the nuclear apex. The features of euspermiogenesis and of the mature eusperm observed in Cerithidea cingulata are similar to many of those in Cerithioideans. The mature acrosome composes of a tapering acrosomal cone, an axial rod and a basal plate. The middle piece of the mature eusperm comprises four equal and non-helical mitochondrial elements around the axonemal microtubules. A dense ring structure separates the middle piece from the glycogen piece. The glycogen piece is the proximal part of the tail which consists of an axoneme surrounded

1M.Sc.(Zoology), Assoc. Prof., Department of Biology, Faculty of Science 2M.Sc.(Biological Science), Depart- ment of Pathology, Faculty of Medicine, Prince of Songkla University, Hat Yai, Songkhla 90112 Thailand Corresponding e-mail: [email protected] Received, 11 April 2003 Accepted, 29 April 2003 Songklanakarin J. Sci. Technol. Euspermatozoon structure and Euspermiogenesis Vol. 25 No. 4 July-Aug. 2003 414 Suwanjarat, J. and Suwaluk, S.

by nine tracts of dense glycogen granules, while the end piece of the tail lacks glycogen. Euspermatozoa and euspermatogenesis of Cerithidea cingulata, though showing some differences between species, do share a number of basic structure features which distinguish potamidid snails from other relatively close families.

Key wards : Cerithidea cingulata, euspermatozoa, Potamididae, spermatogenesis, spermiogenesis

∫∑§—¥¬àÕ ®‘πµ¡“» ÿ«√√≥®√— 1 ·≈– “°≈ ÿ«≈—°…≥å 2 ‚§√ß √â“ß·≈–°“√ √â“ßÕ ÿ®‘¬Ÿ ‡ªÕ√å¡“‚∑´—«¢ÕßÀÕ¬Ω“‡¥’¬« Cerithidea cingulata «. ߢ≈“π§√‘π∑√å «∑∑. 2546 25(4) : 413-422 »÷°…“‚§√ß √â“ß·≈–°“√ √â“ßÕ ÿ®‘™π‘¥¬Ÿ ‡ªÕ√å¡“‚∑´—« (euspermatozoa) ¢ÕßÀÕ¬πÈ”‡§Á¡Ω“‡¥’¬« “¬æ—π∏ÿå Cerithidea cingulata ‚¥¬∫√√¬“¬≈—°…≥–‚§√ß √â“ß‚¥¬≈–‡Õ’¬¥¢Õ߇´≈≈å ◊∫æ—π∏ÿå∑ÿ°√–¬– µ—Èß·µà‡´≈≈å ◊∫æ—π∏ÿå‡√‘Ë¡ µâπ ¬Ÿ ‡ªÕ√å¡“‚∑‚°‡π’¬ (euspermatogonia) ¬Ÿ ‡ªÕ√å¡“‚∑‰´µå (euspermatocyte) ¬Ÿ ‡ªÕ√å¡“µ‘¥ (euspermatid) ·≈–¬Ÿ ‡ªÕ√å¡“‚∑´—« (euspermatozoa) √«¡∑—ÈßÕ∏‘∫“¬¢—ÈπµÕπ°“√‡ª≈’Ë¬π ¿“æ (differentiation) ¢Õ߬Ÿ ‡ªÕ√å¡“µ‘¥ ‡ªìπ¬Ÿ ‡ªÕ√å¡“‚∑´—« ®“°°“√»÷°…“§√—Èßπ’È √ÿª‰¥â«à“ °“√ √â“ßÕ ÿ®‘¢Õß C. cingulata ª√–°Õ∫¥â«¬≈—°…≥–‚§√ß √â“ß æ◊Èπ∞“π§≈⓬§≈÷ß°—∫ÀÕ¬Ω“‡¥’¬«™π‘¥Õ◊ËπÊ „π°≈ÿà¡ Cerithioidean ·≈–Õ ÿ®‘¢Õß C. cingulata · ¥ß≈—°…≥–√à«¡ ¢ÕßÀÕ¬„π«ß§å Potamididae ´÷Ë߇ªìπ≈—°…≥–∑’Ë “¡“√∂®”·π°ÀÕ¬Ω“‡¥’¬««ß§å Potamididae ÕÕ°®“°«ß§å∑’Ë¡’≈”¥—∫ Õπÿ°√¡«‘∏“π„°≈⇧’¬ß°—π‰¥â °“√»÷°…“§√—Èßπ’ȉ¥â√“¬ß“π≈—°…≥–‡©æ“–¢ÕßÀÕ¬™π‘¥π’ȥ⫬

1¿“§«‘™“™’««‘∑¬“ §≥–«‘∑¬“»“ µ√å 2¿“§«‘™“欓∏‘«‘∑¬“ §≥–·æ∑¬»“ µ√å ¡À“«‘∑¬“≈—¬ ߢ≈“π§√‘π∑√å Õ”‡¿ÕÀ“¥„À≠à ®—ßÀ«—¥ ߢ≈“ 90112

Prosobranch sperms have been classified as related to different aspects of reproductive biology typical or atypical depending on whether they are (Franzen, 1955, 1970; Jamieson et al.,1991). Thus, capable of fertilization (Franzen, 1955). These two the spermiogenesis and ultrastructure of the mature types of spermatozoa coexisting in the same species euspermatozoa of gastropods have been studied and in the same individuals. Healy and Jamieson by many investigators (Al-Hajj and Attiga, 1995; (1981) introduced the term euspermatozoa and Attiga and Al-Hajj,1996; Buckland-Nicks and paraspermatozoa to represent the typical and Chia, 1976; Franzen, 1970; Haszprunar, 1988; atypical spermatozoa, respectively. The eusper- Healy, 1983, 1988a, 1988b, 1988c, 1994; Hodson matozoon or typical spermatozoon is filiform and and Bernard, 1989; Morton and Young, 1964; motile. It contains genetic material, and is capable Robertson,1985; Minniti, 1993; Walker and Mac- of fertilization, while the paraspermatozoon or Greogor, 1968). Among these, the ultrastructure of atypical spermatozoon is much larger, lacks mature euspermatozoa and the paraspermatozoa genetic material, and is incapable of fertilization of some species within family Potamididae have (Dohman, 1983; Franzen, 1955; Fretter, 1984; also been described (Healy, 1982a ; 1983, 1986a; Maxwell, 1983). In many caenogastropod, based Healy and Jamieson 1981; Suwanjarat and Klepal, on ultrastructural studies of spermatogenesis and 2001). However, the detailed description of sper- sperm morphology, there are also two types of matogenesis has not been reported at the ultra- sperm present (Healy, 1982a, 1983, 1986a). The structure level for Cerithidea cingulata. use of sperm ultrastructure for phylogeny and Cerithidea cingulata (Gmelin, 1791) or the is now widely accepted and has been Girdled Horn Shell is a brackish small shelled Songklanakarin J. Sci. Technol. Euspermatozoon structure and Euspermiogenesis Vol. 25 No. 4 July-Aug. 2003 415 Suwanjarat, J. and Suwaluk, S. gastropod of the family Potamididae. Despite its centrally located nucleus with small clumps of role in the important food chain of the electron-dense chromatin. One or two nucleoli are ecosystem, the population size of Cerithidea also often found. The nuclear envelope is electron- cingulata is an indicator of environmental pollu- dense, the cytoplasm contains numerous ribosomes, tion. In Pattani Bay, the Southern Gulf of Thailand, a Golgi body, endoplasmic reticulum, and a few where Cerithidea cingulata is abundant, molluscs small oval or round mitochondria (Figure 1). are used to monitor the level of pollution in coastal waters (Kan-atireklap et al, 1997 and Swennen Spermatocytes et al, 2001). Thus, the study of the reproductive The eccentric nucleus of spermatocytes is biology of this species is essential. The present smaller than that of spermatogonia. The nuclear work studies the spermiogenesis and the mor- chromatin is distributed as small granules through- phology of mature euspermatozoa in Cerithidea out the nucleus. The cytoplasmic organelles are cingulata (Gmelin, 1791) that inhabit the muddy abundant with a small Golgi body, endoplasmic sands in the intertidal zone close to the mangrove reticulum, and a number of round moderate sized forests of the western coast of Southern Thailand. mitochondria. Some paraspermatocytes or the This study not only provides useful information atypical spermatocytes are found among sper- on the reproductive biology but also helps deter- matocytes (Figure 2). The paraspermatocyte is mining the taxonomic relationship between many characterized by a large electron dense granule other small shelled Cerithioideans. and the many dilated vesicles of the Golgi body, and the increased endoplasmic reticulum filling Materials and Methods the entire cytoplasm. The primary and secondary spermatocytes are often difficult to distinguish. Mature specimens of Cerithidea cingulata The cytoplasm of the late spermatocyte contains were collected from March-May 2001 in the inter- a smooth and rough endoplasmic reticulum. The tidal of the mangrove forest along the Golgi complex is generally arranged in flattened coast of Satun province, Southern Thailand. The cisternae close to the nucleus with more or less live specimens were taken to the laboratory of the dilated vesicles in the peripheral regions of the department of Biology, Prince of Songkla Univer- cytoplasm. Some mitochondria are found scatter- sity, Hat Yai. Small pieces of testes were fixed in ing within the cytoplasm. Most developing sper- 4% paraformaldehyde phosphate buffer pH 7.3 at matocytes are connected by intercellular bridge 4ºC, washed in phosphate buffer, postfixed in 1% (Figure 3). osmium tetroxide for 1 hr, stained for 45 min in 2% uranyl acetate, dehydrated in an ethanol series Spermiogenesis and embedded in Epon 812. Semithin and ultrathin During spermiogenesis, the spermatids sections were cut with a ultramicrotome model undergo differentiation to become mature sperma- MTXL, the ultrathin sections were collected on 200 tozoa. The following major steps then take place: mesh copper grids, stained with uranyl acetate and nucleus condensation; acrosome formation; mid- lead citrate. The sections were examined with a piece development. These phenomena are des- transmission electron microscope Zeiss EM 9S-2, cribed as follows: at the Ultrastructure Research Unit, Institute of Nuclear condensation: The nucleus of Zoology, University of Vienna, Austria. spermatid prior to spermiogenesis is spherical, eccentric with no apparent nucleolus. The cyto- Results plasm of the early spermatid is characterized by a prominent well developed Golgi body and the Spermatogonia rough endoplasmic reticulum is prominent in the The spermatogonia possess a large round, cytoplasm between the two daughter cells (Figure Songklanakarin J. Sci. Technol. Euspermatozoon structure and Euspermiogenesis Vol. 25 No. 4 July-Aug. 2003 416 Suwanjarat, J. and Suwaluk, S.

Figure 1. Spermatogonia showing round nucleus (N) contains one or two nucleoli (nu). Scale bar = 4 µm. Figure 2. Primary spermatocytes (scy) and paraspermatocytes (pas). N: nucleus; m: mito- chondria. Scale bar = 6 µm Figure 3. Late spermatocyte connected by a cytoplasmic bridge. Note the Golgi body (Gb) with the flattened cisternae. m: mitochondria. Scale bar = 3 µm Figure 4. Early spermatid with the thickening of the nuclear chromatin. Gb: Golgi body; m: mitochondria; N: nucleus; rER: rough endoplasmic reticulum. Scale bar = 3 µm Figure 5. Early spermatid showing the gathering enlarged mitochondria at the nuclear base (arrow head). m: mitochondria; N: nucleus. Scale bar = 1.5 µm Figure 6. Spermatid showing beginning of chromatin condensation. N: nucleus; m: mito- chondria. Scale bar = 3 µm Songklanakarin J. Sci. Technol. Euspermatozoon structure and Euspermiogenesis Vol. 25 No. 4 July-Aug. 2003 417 Suwanjarat, J. and Suwaluk, S.

4). The large mitochondria developed by the fusion at the posterior nuclear pole (Figures 8-10). Then, of the small ones, are clustered close together at the nucleus begins to expand laterally (Figure 11). the presumptive posterior pole of the nucleus During the middle spermatid stage the nuclear (Figure 5). The chromatin granules of this stage chromatin transforms from a dispersed state to fine are condensed at the periphery of the nucleus, form- fibrillar threads and latterly to thick fibrillar threads, ing the thick layer beneath the nuclear envelope which are elongated and orientated longitudinally (Figure 6). The posterior nuclear pole is marked along the nuclear vertical axis. At the next step, by the attachment of large spherical mitochondria the chromatin fibrils thicken to form lamellae and the implantation fossa with the axoneme (Figures 12-13). During further development the insertion (Figure 7). As spermiogenesis proceeds, nucleus lengthens and the chromatin lamellae the granular nuclear chromatin which has been continue to become thicker (Figure 14), coupled thickening at the periphery begins to accumulate with axonemal growth, and eventually increase the

Figure 7. Details of mitochondrial spheres surrounding the axoneme (ax). N: nucleus; m: mitochondria. Scale bar = 1.5 µm Figure 8-9. Spermatid showing nucleus with the accumulating of granular chromatin and attachment of mitochondrial spheres. Gb: Golgi body; m: mitochondria; N: nucleus; pav: proacrosomal vesicle. Scale bar = 4 µm Figure 10. Spermatid showing the Golgi body (Gb). N: nucleus; m: mitochondria. Scale bar = 2.5 µm Songklanakarin J. Sci. Technol. Euspermatozoon structure and Euspermiogenesis Vol. 25 No. 4 July-Aug. 2003 418 Suwanjarat, J. and Suwaluk, S. nuclear length. Toward the late spermatid stage, a glycogen piece and an endpiece. The nucleus of the nuclear chromatin continues to condense and mature spermatozoa is elongated with homo- finally the nucleus appears homogeneous and geneous electron dense chromatin, the nucleus has electron dense. a short posterior invagination, accommodating the Acrosome formation: In the earliest stage of proximal portion of the axoneme. The apex of the acrosomal development, electron-dense granules nucleus is situated by the tapering acrosome which arising from the Golgi apparatus are obvious in comprises of an acrosomal cone, the acrosomal rod the cytoplasm of the early spermatid (Figure 8). or axial rod, and the basal plate. Behind the nucleus This structure form the proacrosomal vesicle. The is the middle piece with an electron-dense plate or proacrosomal vesicle elongates and differentiates midpiece flange structure at the junction (Figure into pre-attachment acrosome. This pre-attachment 18). The midpiece of the euspermatozoon is acrosome moves anterioly towards an apical characterized by the four equal and non-helical depression of the condensing fibrous phase of the mitochondrial elements around the axonemal nucleus (Figure 13), then the large dense granule microtubules. Each mitochondrial element is con- attaches to the nuclear apex, and flattens to form structed of multiple, curved, paralleled cristal plates a basal plate. The basal plate with the acrosomal (Figure 16). There is a circular dense band around cone placed upon is set in the apical depression of the axoneme separating the mitochondrial portion the lamella phase of the nucleus (Figure 14). The or midpiece from the glycogen piece (Figure 16). body of the acrosome begins to elongate, and the The proximal part of the tail or the glycogen piece dense internal supporting structures appear within consists of an axoneme surrounded by the tracts of it (Figure 14). In the semimature sperm, the dense glycogen granules, while the distal region or acrosomal cone fuses over the anterior half of its the end piece of the tail lacks glycogen (Figure 17). length, thus constricting the subacrosomal space The transverse section of glycogen piece shows with an acrosomal rod arising in its center (Figure the 9+2 pattern of microtubular arrangement 18). surrounded by a sheath of nine tract glycogen Midpiece development: The development granules limited by the plasma membrane (Figure of the middle piece is closely associated with 19). mitochondria and centrioles. In the early spermatid, the mitochondria which are distributed throughout Discussion the cytoplasm begin to aggregate at the developing posterior pole of the condensing nucleus to form The Cerithidea cingulata reproduces using the four large spherical mitochondria. These internal fertilization and its sperm shows the mitochondria cluster around the proximal region characteristic features of modified euspermatozoa of the axoneme which is initially inserted into the of caenogastropods as described by Maxwell implantation fossa at the base of the nucleus (Figure (1983) and Voltzow (1994). The euspermatogenesis 7). The elongation of the four giant mitochondria and mature sperm of Cerithidea cingulata includes was obvious in the cytoplasm of the mid spermatid many common features that were reported in all (Figure 15), and was eventually evident in the form other cerithioidians (Attiga and Al-Hajj, 1996; of the four mature midpiece elements surrounding Healy, 1982a, 1982b, 1983, 1986a, 1986b, 1994; the axoneme (Figure 16). Suwanjarat and Klepal, 2001). The ultrastructure of Cerithidea cingulata mature sperm has been Mature euspermatozoa described by Healy (1983) and confirmed herein The mature euspermatozoon of Cerithidea with more details on spermatogenesis. As in cingulata is a filiform cell, consisting of an acro- numerous species studied (Buckland-Nicks and some and a nucleus as a head region; a midpiece Chia, 1976; Hodson and Bernard, 1989), the sper- region; and a tail region which is subdivided into matogonia of Cerithidea cingulata possess a large Songklanakarin J. Sci. Technol. Euspermatozoon structure and Euspermiogenesis Vol. 25 No. 4 July-Aug. 2003 419 Suwanjarat, J. and Suwaluk, S.

Figure 11. Spermatid with the antero-posteriorly compressed nucleus (N) and enlarged mitochondrial spheres (m). Gb: Golgi body. Scale bar = 4 µm Figure 12. Transverse section of spermatid showing the lamella nucleus (N). ac: acrosome; ax: axoneme; m: mitochondria. Scale bar = 2 µm Figure 13. Spermatid shows the longitudinal arrangement of the thickening fibrils in the nucleus and the elongated acrosome. ac: acrosomal cone; bp: basal plate; N: nucleus; ss: subacrosomal space. Scale bar = 2 µm Figure 14. Transverse section through elongated acrosome. Note the invagination in the basal pole of the nucleus (n). ax: axoneme. Scale bar = 2 µm Figure 15. Spermatid showing longitudinal section of midpiece (mp). Scale bar = 1.5 µm Figure 16. Longitudinal section through midpiece (m) and gylcogen piece (gy) showing dense struc- ture (arrow) at the junction. ax: axoneme; m: mitochondria. Inset: cross section of mito- chondrial elements. Scale bar = 0.6 µm Figure 17. Longitudinal section through glycogen piece (gy) and tail. Note the junction of glycogen piece and end piece (arrow head). ax: axoneme; ep: end piece. Scale bar = 0.5 µm Figure 18. Longitudinal section through head region of euspermatozoon. Note the electron dense plate (arrow). ac: acrosome; ar: axial rod; n: nucleus. Scale bar = 1 µm Figure 19. Transverse section through glycogen piece (gy). Scale bar = 0.5 µm Songklanakarin J. Sci. Technol. Euspermatozoon structure and Euspermiogenesis Vol. 25 No. 4 July-Aug. 2003 420 Suwanjarat, J. and Suwaluk, S. round nucleus with one or two nuclei and the The association between the Golgi complex and the cytoplasmic organelles are poorly developed. developing acrosome is clearly seen in Cerithidea In early spermatids, the presence of a well cingulata. Moreover, the appearance of the inter- developed Golgi complex suggested that it could nal and external supporting structure within the possibly be involved in acrosome and midpiece acrosomal cone during development is similar to development during spermiogenesis. Many of the many species of such as: spermiogenesis features observed in Cerithidea caeruleum (Al-Hajj and Attiga,1995), cingulta appear to be common for caenogastropods bifasciata and Clypeomorus tuberculatus (Attiga in general. These include substructural changes in and Al-Hajj, 1996), and in some other Caeno- the nucleus. In Cerithidea cingulata, the pattern of gastropods (Buckland-Nicks and Chia, 1976). This nuclear condensation passing through the granular, structure is supposed to be microtubules that fibrillar and lamellar phase is similar to Cerithidea provide the frame and shaping of acrosomal cone. obtusa (Suwanjarat and Klepal, 2001), as well as Healy (1983) divided Potamididae into two sub- to those reported in other prosobranchs (Al-Hajj groups due to the pronounced differences on the and Attiga, 1995; Attiga and Al-Hajj, 1996; Buck- basis of euspermatozoon midpiece and acrosome land-Nicks and Chia, 1976; Healy, 1982b, 1986a structure, existing between the two subfamilies of 1986b, 1988b; Walker and MacGregor, 1968). the potamididae. Cerithidea cingulata has been However, this slightly differs from P. ebeninus, classified into the subgroup that the acrosomal cone the potamidid snail that the early condensation is is elongated and the midpiece composed of four reticular (Healy, 1982a). Mature eusperm ultra- non-helical equaled size midpiece elements. The strucure of Cerithidea cingulata basically consists non-helical arrangement of mitochondrial elements of a rod shaped nucleus with posteriorly invagina- around the axoneme which are found in Cerithidea tion, which according to Healy (1983) is standard cingulata are considered a primitive structure com- to all Potamidid spermatozoa. pared to the helical mitochondrial sheath seen in In Potamididae, the acrosomal cone is other mesogastropods and neogastropods (Giusti, elongate and it varies in shape from truly conical 1969; Walker and MacGregor,1968). The midpiece to flat. In Cerithidea cingulata, the structure of the elements of Cerithidea cingulata mitochondria mature acrosome in general, is similar to those of show parallel crystal plates which are a common other mesogastropod and neogastropod eusper- feature of the superfamily (Healy, matozoa which consist of an acrosomal cone, axial 1983). The process of forming the glycogen piece rod, and basal plate. However, species-specific was not traced, the glycogen piece of mature features of the acrosome have been seen with Cerithidea cingulata euspermatozoa does not differ comparison of Cerithidea obtusa. The axial rod of in any respect from the configuration shown to exist the mature acrosome in Cerithidea cingulata is in many other Caenogastropod (Healy, 1983). clearly seen and the acrosomal cone is oval in the In conclusion, euspermatozoa and eusper- basal region and was become flattened further matogenesis of Cerithidea cingulata, though anteriorly, while the acrosomal cone of Cerithidea showing some differences between species, do obtusa is less flattened or oval-shaped anteriorly share a number of basic structural features which in transverse section and the axial rod is absent distinguish potamidid snails from other relatively (Suwanjarat and Klepal, 2001). The acrosomal close families likes cerithiidid snails. Although the development in Cerithidea cingulata is consistent early stages of paraspermatozoa have been seen with that observed in Cerithidea obtusa of no evidence of mature paraspermatozoa could be Potamididae (Suwanjarat and Klepal, 2001), found. In order to obtain the rudimentary inform- and Clypeomorus tuber- ation of Cerithidea cingulata, the further study on culatus of Cerithiidae (Attiga and Al-Hajj,1996). parasperm of this species should be conducted. Songklanakarin J. Sci. Technol. Euspermatozoon structure and Euspermiogenesis Vol. 25 No. 4 July-Aug. 2003 421 Suwanjarat, J. and Suwaluk, S.

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