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Comparative ultrastructural study of spermatozoa in some oyster species from the Asian-Pacific Coast
Micron 43 (2012) 365–373
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Comparative ultrastructural study of spermatozoa in some oyster species from the Asian-Pacific Coast Olga V. Yurchenko A.V. Zhirmunsky Institute of Marine Biology, Far Eastern Branch of Russian Academy of Science, Palchevsky Street, 17, Vladivostok 690041, Russia
a r t i c l e
i n f o
Article history: Received 16 August 2011 Received in revised form 26 September 2011 Accepted 26 September 2011 Keywords: Crassostrea cf. rivularis Crassostrea nippona Saccostrea cf. mordax Reproduction Sperm ultramorphology
a b s t r a c t Sperm organization in the oysters Crassostrea gigas, Crassostrea nippona, Crassostrea cf. rivularis and Saccostrea cf. mordax inhabiting Asian Pacific coast was studied. The spermatozoa of all studied species had a number of common morphological characters such as a cup-like acrosome with heterogeneous matrix on its top, an axial rod in the subacrosomal space, a barrel-shaped nucleus, four mitochondria in the midpiece, pericentriolar complexes, and a 9 + 2-organized flagellum. The spermatozoa of C. cf. rivularis differed from the other species by having cytoplasm processes in the midpiece region. Such structures have never been described in the Ostreidae. Additionally, each species could be identified by the shape and size of sperm compartments (acrosome, nucleus, anterior nuclear fossa). The most significant interspecific difference was found in the size of an anterior nuclear fossa. The smallest anterior nuclear fossa was found in C. cf. rivularis (about 0.24 m in length reaching about 22% of the nuclear length) while the biggest in C. gigas from the Sea of Japan (about 0.53 m in length reaching about 46% of the nuclear length). The spermatozoa of C. gigas collected from the Sea of Japan and Taiwan Strait differed significantly in almost all the studied parameters. Since sperm morphology has been successfully used for species differentiation, this suggests the existence of two species rather than two populations. The data obtained indicate the species-specific difference in the sperm ultrastructure within the Ostreidae, which may be identified both ultrastructurally and morphometrically. © 2011 Elsevier Ltd. All rights reserved.
1. Introduction Sperm morphology is believed to have useful application for taxonomic and phylogenetic analysis specifically at or above the family level (see Popham, 1979; Healy, 1996; Kafanov and Drozdov, 1998). But in Franzen’s (1983) opinion, there are not even two species of bivalve mollusk with identical spermatozoa. A number of studies have been conducted of closely related species. They show that even if the spermatozoa of these species are rather similar in their ultramorphology, they still can be recognized by morphometrical analysis or with cytochemical staining (genera Solen – Hodgson et al., 1987; Donax – Hodgson et al., 1990; Bathymodiolus – Eckelbarger and Young, 1999; Tegillarca – Yang and Zhu, 2011). Similar studies were conducted using mollusks of other taxa (genera Littorina – Sousa and Azevedo, 1988; Haliotis – see review by Healy et al., 1998; Brachidontes – Introíni et al., 2004; Fissurella – Collado and Brown, 2006; Tegula – Collado et al., 2008). To date the sperm structure has been studied in 5 oyster species among about 40 species of the family Ostreidae from all over the world: Saccostrea commercialis (Healy and Lester, 1991), Ostrea edulis (Sousa and Oliveira, 1994), Crassostrea virginica (Daniels et al.,
E-mail address: [email protected] 0968-4328/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2011.09.017
1971; Eckelbarger and Davis, 1996), Crassostrea angulata (Sousa and Oliveira, 1994), Crassostrea gigas (Bozzo et al., 1993, 2008; Komaru et al., 1994; Gwo et al., 1996; Dong et al., 2005; Drozdov et al., 2009; Yurchenko et al., 2010). According to the available publications, there are two points of view on the species-specificity of oyster sperm. On the one hand, the spermatozoa of the studied species or even genera are very similar (for example, C. angulata and O. edulis) (Sousa and Oliveira, 1994). On the other hand the size and shape of the nucleus and acrosome may be species-specific (Gwo et al., 1996). To verify both points of view the present work was carried out. This paper studies the sperm ultrastructure and linear sizes of sperm head components in four species inhabiting the Asian Pacific coast (C. gigas, Crassostrea nippona, Crassostrea cf. rivularis, Saccostrea cf. mordax). Since our preliminary observations have indicated some dimensional difference in the sperm sizes between two populations of the pacific oyster C. gigas from Taiwan Strait and the Sea of Japan, the specimens from both populations were included into the list of studied oysters for detail investigation. 2. Materials and methods Oysters from four regions of the Asian Pacific coast were collected by SCUBA equipment. Some characters of individuals and
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Table 1 Characteristics of sampling sites and collected individuals. Oyster species
Sampling site
Date of sampling
Sample size (n)
Size of individuals (cm)
Crassostrea gigas (1)
Inner part of Vostok Bay, Peter the Great Bay of the Sea of Japan 42◦ 53 N 132◦ 44 E Korea Strait, Busan City coast (no precise coordinates) Tsaiyuan shallow water on Penghu Island, Penghu County in south-western Taiwan 23◦ 31 N 119◦ 33 E Nha Trang Bay, the South China Sea 12◦ 17 N 109◦ 14 E Nha Trang Bay, the South China Sea 12◦ 13 N 109◦ 13 E
June, 2006
5
10–15
August, 2009
3
20–22
May, 2006
5
10–15
March, 2009
5
7–9
June, 2010
3
4–5
Crassostrea nippona Crassostrea gigas (2)
Crassostrea cf. rivularis (Fig. 1A) Saccostrea cf. mordax (Fig. 1B)
sampling sites are shown in Table 1. The shell morphology of nondescript oyster species is presented in Fig. 1. The oysters were opened and their sex was identified after preparing gonad smears on object slides. Only males from each site were studied. Pieces of gonad tissues were fixed in 2.5% glutaraldehyde (Sigma) in 0.1 M cacodylate buffer (pH 7.5), with 21 mg/ml NaCl added, for 2 h at 4 ◦ C. The specimens were then rinsed several times in buffer, and transported to the laboratory of the Institute of Marine Biology (Vladivostok, Russia) at a temperature of +4 ◦ C. In the laboratory the material was post-fixed for 2 h with 2% osmium tetroxide prepared in the same buffer and then washed in buffer before dehydration. After dehydration in a graded ethanol series and acetone, the specimens were embedded in Spurr resin (Spurr,
EMS). The ultrathin sections (50–60 nm) were made using ultramicrotome Leica UC6 and stained with 2% aqueous uranyl acetate and lead citrate, and viewed with JEOL JEM 100S transmission electron microscope. To estimate the sizes of some sperm compartments (acrosome, nucleus and anterior nuclear fossa [Fig. 2]) 30 electronograms were made per species (10 for a male specimen, three males of each species) with spermatozoa in straight longitudinal projection (axial rod extended from basal part of nuclear fossa to acrosome). The width of acrosome was measured at the basal diameter and the width of the anterior nuclear fossa at the widest part. The results were expressed as the mean ± SD. The species were compared using Student‘s t-test at a significance level of 0.05.
Fig. 1. Oyster species from Nha Trang Bay (Vietnam). Left and right valves, external and internal views. (A) Crassostrea cf. rivularis and (B) Saccostrea cf. mordax.
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situated (Fig. 3 line C, C. cf. rivularis). They have also been recorded in transverse sections through the mitochondrial region (Fig. 3 line D, C. cf. rivularis). Flagellums have common 9 + 2 microtubule organization. 3.2. Dimensional characters
Fig. 2. Sperm organization in oysters. Scheme for measuring sperm compartments by electron microscopy.
3. Results 3.1. Ultramorphology The spermatozoa of all studied species consist of an acrosomal complex, a nucleus, a midpiece and a flagellum. Each nucleus is barrel-shaped, with granular electron dense chromatin; anterior and posterior fossae are located, respectively on the apical and basal parts of the nucleus (Figs. 3 line A and 4A). In all presented species the acrosomal complex includes an acrosome and a subacrosomal space full of flaky substance with moderate electron-density. The contents of the acrosome differentiate into a large and homogeneous basal region and a small heterogeneous apical region with laminar structure (Figs. 3 line B and 4B). The subacrosomal space is formed by the invaginated basal part of the acrosome and an anterior nuclear fossa and contains an axial rod extended from the basal part of the anterior nuclear fossa to the apical part of the subacrosomal space (Figs. 3 line B and 4B). The anterior nuclear fossa has different shapes in the studied species. In C. cf. rivularis it looks like a small depression, in S. cf. mordax it is deep and cylindrical (Fig. 4A), in C. gigas from the Japan Sea and Taiwan populations and in C. nippona cup-like (Fig. 3 line A). This visual difference is confirmed by morphometric analysis (see Section 3.2). Spermatozoa with an overacrosomal electron-lucent terminal knob were found in all studied species. The midpiece of spermatozoa consists of four mitochondria surrounding a centriolar complex (Figs. 3 line C and 4C). The proximal and distal centrioles orientated orthogonally to each other have accessory structures. The proximal centriole is connected to a short centriolar rootlet present as a conical mass and extended into the posterior nuclear fossa (Figs. 3 line D and 4D). The distal centriole has nine radially arranged satellite fibers attaching it to the plasma membrane (Figs. 3 line E and 4E). Among the studied species only C. cf. rivularis has a specific midpiece organization. In the mitochondrial region rather long cytoplasmic processes, about 1 m, are
Despite rather similar sperm ultramorphology, spermatozoa have species-specific linear dimensions of the nucleus, acrosome and anterior nuclear fossa (Table 2). For interspecific comparison, it seems reasonable to compare both linear sizes and linear ratios: length/width ratios of sperm compartments (for example, nucleus length to its width) and length ratios between sperm compartments (for example, acrosome length to nucleus length, etc.). Among the species the length/width ratio of the nuclei in the spermatozoa varies from 0.54 in C. gigas from Taiwan Strait to 0.69 in S. cf. mordax (Table 2); the difference was statistically significant both between these species and in comparison with other species (Fig. 5A and Table 3). The spermatozoa of Taiwan C. gigas are characterized by a shortened and wide acrosome (length/width ratio 0.43 vs 0.49–0.53 in the other species) (Fig. 5B). The ratio of acrosome length to nucleus length indicates that spermatozoa in S. cf. mordax and C. cf. rivularis have short acrosomes (Fig. 5C) constituting about 34–35% of nucleus length, while in C. gigas from both populations and C. nippona the acrosome is nearly half as long as the nucleus (Table 2). Only C. nippona and C. gigas from Taiwan Strait were almost similar in this parameter (Fig. 5C and Table 3). According to measuring, the spermatozoa of the studied species vary in the shapes and sizes of the anterior nuclear fossa (Table 2). S. cf. mordax is the only species with longer than wide anterior nuclear fossa (Table 2). In the other species the width was significantly bigger than the length (Table 2 and Fig. 5D). Although the linear sizes of C. nippona and C. cf. rivularis are notably unequal, the length/width ratios of the fossa do not show any significant difference (Table 3). The ratio of fossa length to nucleus length significantly varies among almost all the studied species (Fig. 5E) with the exception of S. cf. mordax, in which this parameter is similar to that of C. gigas from Taiwan Strait (Table 3). Interspecies difference in the ratio of anterior fossa width to nucleus width is less pronounced (Fig. 5F), and the anterior nuclear fossa occupies about 33–35% of the nucleus width excluding C. cf. rivularis and S. cf. mordax with rather compact nuclear fossae (23% and 24%, respectively). 4. Discussion 4.1. Ultrastructural characteristics The comparison of sperm structure in four species of Ostreidae shows that they closely resemble one another and those in other investigated ostreids (S. commercialis – Healy and Lester, 1991, O. edulis – Sousa and Oliveira, 1994, C. virginica – Daniels et al., 1971; Eckelbarger and Davis, 1996, C. angulata – Sousa and Oliveira, 1994, C. gigas – Bozzo et al., 1993; Komaru et al., 1994; Gwo et al., 1996; Dong et al., 2005; Drozdov et al., 2009; Yurchenko et al., 2010). However, the present study reveals some ultrastructural features that have not been described earlier. In the species studied here the electron-lucent terminal knob was often noticed in the tip of the acrosome beneath the cytoplasmic membrane. Since such a structure has earlier been found in C. gigas from the Sea of Japan but was not recorded in the C. gigas from other regions, it can be interpreted as different degrees of spermatozoon maturity rather than a “possible species-specific feature” (Yurchenko et al., 2010). In addition, the present wider study indicates that this knob can be found in other oyster species and cannot be accepted as an individual trait of C. gigas.
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Fig. 3. Sperm ultramorphology in the four oysters belonging to the genus Crassostrea. (See the species names on the top of the figure.) (Line A) Longitudinal section through the spermatozoon. (Line B) Longitudinal section through the acrosome and subacrosomal space (asterisk). (Line C) Transverse section through the midpiece. (Line D) Longitudinal section through the midpiece. (Line E) Section through the pericentriolar complex of the distal centriole. Abbreviations: a, acrosome; anf, anterior nuclear fossa; ar, axial rod; av, acrosomal vesicle; cp, cytoplasmic process; dc, distal centriole; f, flagellum; m, mitochondrion; n, nucleus; pc, proximal centriole; pnf, posterior nuclear fossa; r, rootlet; sf, satellite fibers; tk, terminal knob. Scale bars: (Line A) 1 m; (Line B) 0.3 m; (Lines C and D) 0.5 m and (Line E) 0.2 m.
Table 2 Summary of dimensions (m) of spermatozoa compartments in the family Ostreidae. Organelles
Crassostrea cf. rivularis Genus Saccostrea Saccostrea cf. mordax Saccostrea commercialis Genus Ostrea Ostrea edulis
a
Nucleus
Anterior nuclear fossa
Acrosome length/ nucleus length
Fossa length/ nucleus length
Fossa width/ nucleus width
Source
Length
Width
L/W
Length
Width
L/W
Length
Width
L/W
0.58 ± 0.05
1.09 ± 0.11
0.53 ± 0.05
1.15 ± 0.09
1.90 ± 0.19
0.61 ± 0.06
0.53 ± 0.08
0.62 ± 0.08
0.87 ± 0.15
0.50 ± 0.05
0.46 ± 0.07
0.33 ± 0.03
Present study
0.49 ± 0.05
1.13 ± 0.11
0.43 ± 0.03
1.07 ± 0.09
1.99 ± 0.19
0.54 ± 0.04
0.43 ± 0.06
0.67 ± 0.09
0.65 ± 0.11
0.46 ± 0.04
0.40 ± 0.05
0.33 ± 0.02
Present study
0.43 ± 0.06
0.85 ± 0.07
0.5
0.96 ± 0.08
1.55 ± 0.12
0.62
–
–
–
0.45
–
–
0.53 ± 0.07
1.02 ± 0.11
0.52 ± 0.05
1.12 ± 0.09
1.83 ± 0.17
0.61 ± 0.05
0.35 ± 0.04
0.64 ± 0.06
0.55 ± 0.09
0.47 ± 0.04
0.31 ± 0.05
0.35 ± 0.02
0.5
1.1
0.45
1.0
–
–
–
–
–
–
–
–
0.38 ± 0.05
0.74 ± 0.08
0.52 ± 0.04
1.09 ± 0.11
1.74 ± 0.16
0.62 ± 0.07
0.24 ± 0.04
0.41 ± 0.06
0.58 ± 0.09
0.35 ± 0.04
0.22 ± 0.04
0.24 ± 0.02
Gwo et al. (1996) Present study Sousa and Oliveira (1994) Present study
0.39 ± 0.05
0.8 ± 0.09
0.49 ± 0.04
1.17 ± 0.15
1.71 ± 0.19
0.69 ± 0.07
0.5 ± 0.09
0.39 ± 0.05
1.3 ± 0.22
0.34 ± 0.03
0.43 ± 0.05
0.23 ± 0.02
0.2
0.8–1.0
0.22
1.0
1.4–1.5
0.69
0.4–0.5
–
–
0.2
0.45
–
0.5
0.9
0.56
–
–
–
–
–
–
–
–
–
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Genus Crassostrea Crassostrea gigas (Sea of Japan) Crassostrea gigas (Taiwan Strait) Crassostrea gigas Crassostrea nippona Crassostrea angulata
Acrosomea
Present study Healy and Lester (1991) Sousa and Oliveira (1994)
The size includes the length of an electron-dense granule without an electron-lucent knob.
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An anterior banded substructure found both in the species described here and in the previously studied ones (aka “whorl-like component” [Brandriff et al., 1978] or “laminar-like portion of acrosomal vesicle” in C. gigas [Bozzo et al., 1993], “semicircular dense laminae” in C. angulata and O. edulis [Sousa and Oliveira, 1994] and “series of transverse bands” in S. commercialis [Healy and Lester, 1991]) seems to be an attribute of the family Ostreidea. It should be noted that in the previous reports the fixation procedure varied in the composition and concentration of primary fixers (from 2% to 5% glutaraldehyde or mixture of 2% paraformaldehyde and 2.5% glutaraldehyde prepared in cacodylate or phosphate buffers with the addition of NaCl or sucrose or van’t Hoff’s artificial sea water), as well as in the composition and concentration of staining solution of uranyl acetate (from 2% to 5% of aqueous or alcoholic solution); in some cases sections were treated with lead citrate only. Analysis of morphological difference in sperm ultrastructure made by Healy et al. (1998) showed that depending on fixation, cytochemical treatments and section staining methods employed, the same acrosomal components in the sperm of Haliotidae species may appear either more or less electron dense. Therefore, C. virginica, which has typical acrosome morphology but lacks the anterior banded substructure (Eckelbarger and Davis, 1996), is probably not an exception, and future reinvestigation using other fixers and subsequent treatment procedures may demonstrate that its acrosome has the similar fine organization. The presence of cytoplasmic processes in the midpiece region of spermatozoa is a species-specific trait of C. cf. rivularis. Among the mollusk species, the cytoplasmic structures in the midpiece of spermatozoa called “collar” or “membranous skirts” were noticed in some species of Patellogastropoda (see Hodgson and Chia, 1993) and Cephalopoda (see Healy, 1996). Their occurrence is likely to be connected with aspects of fertilization process, because spermatozoa with membranous skirt among Cephalopoda were attributed to species with fertilization within the mantle cavity (see Healy, 1996). The oysters of Ostrea and Lopha genera have a similar way of fertilization and are larviparous, while the oysters of Crassostrea and Saccostrea genera discharge their eggs and sperm directly into the surrounding seawater (Buroker, 1985; Foighil and Taylor, 2000). It is expectable, that Ostrea species could have sperm with accessory structures helping it to penetrate a female organism and attach and/or anchor to egg surface under stream conditions. But the organization of O. edulis spermatozoa does not show any accessories (Sousa and Oliveira, 1994). According to Asif (1979), C. rivularis is an oviparous species therefore the role of the cytoplasmic processes is related to some other aspect of reproductive biology and will have to be the subject of further discussion. 4.2. Dimensional characteristics In addition to morphological traits, the dimensional measurements of sperm components also demonstrate interspecies differences, which confirm the suggestion of Gwo et al. (1996) about the distinctions of linear sizes of acrosome and nucleus in ostreid species. Moreover, the acrosome/nucleus ratios underline the individuality of sperm organization in some species of the Ostreidae (Fig. 5). For example, C. gigas from Taiwan Strait differs from the others by a lower acrosome/nucleus length ratio, which makes the shape of sperm head more spherical. The spermatozoa of C. cf. rivularis and S. cf. mordax are distinguished among other studied species by a rather compact acrosome. In Table 2 the information
Fig. 4. Sperm ultramorphology of Saccostrea cf. mordax. (A) Longitudinal section through the spermatozoon. (B) Longitudinal section through the acrosome and subacrosomal space (asterisk). (C) Transverse section through the midpiece. (D) Longitudinal section through the midpiece. (E) Section through the pericentriolar
complex of the distal centriole. Abbreviations: a, acrosome; anf, anterior nuclear fossa; ar, axial rod; av, acrosomal vesicle; dc, distal centriole; m, mitochondrion; n, nucleus; pc, proximal centriole; pnf, posterior nuclear fossa; r, rootlet; sf, satellite fibers. Scale bars: (A) 1 m; (B) 0.3 m; (C and D) 0.5 m and (E) 0.2 m.
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Fig. 5. Whisker plots showing species-specific differences in the coefficients of sperm compartments ratios. Ratios of (A) nucleus length to nucleus width, (B) acrosome length to acrosome width, (C) acrosome length to nucleus length, (D) anterior nuclear fossa length to its width, (E) anterior nuclear fossa length to nucleus length and (F) anterior nuclear fossa width to nucleus width. X-axis shows: 1, Crassostrea gigas from the Sea of Japan; 2, C. nippona; 3, C. gigas from Taiwan Strait; 4, C. cf. rivularis and 5, Saccostrea cf. mordax.
on the size limits of the sperm compartments in the three genera of Ostreidae family is summarized. Since the dimensional data of species described in previous reports are rather scanty, it is difficult to single out some definite dimensional features that would be characteristic for the each genus; nevertheless, the suggestion
about different sizes of sperm compartments can be confirmed for the studied oysters inhabiting Asian coast. Along with nuclear and acrosomal sizes, the size of the anterior nuclear fossa may also be accepted as a vivid individual character in the studied species. Apparently, the nuclear fossa can occupy up
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Table 3 Significance values of pairwise test for the interspecific comparison of sperm compartments.
C. cf. rivularis
C. nippona
C. gigas (Taiwan Strait)
C. gigas (Sea of Japan)
Ratio
C. gigas (Sea of Japan)
NL/NW AL/NL AL/AW FL/NL FL/FW FW/NW NL/NW AL/NL AL/AW FL/NL FL/FW FW/NW NL/NW AL/NL AL/AW FL/NL FL/FW FW/NW NL/NW AL/NL AL/AW FL/NL FL/FW FW/NW
a
**
***
a
a
**
***
***
***
***
C. cf. rivularis
C. nippona
***
Saccostrea cf. mordax
morphological and molecular methods should be conducted to clarify its taxonomic position. Summing up the results, the ultrastructure and morphometrical analysis showed that each of the studied species had its unique sperm organization. Therefore, the mollusks from the family Ostreidae do not refute but confirm the Franzèn’s (1983) statement about presence of species-specific spermatozoa in bivalve mollusks. But whether described morphological and morphometrical features of spermatozoa involve into formation of interspecies reproductive barrier is required future experimental studies.
a
a
a
***
*
***
***
a
a
*
***
***
***
***
a
***
**
***
***
***
***
***
***
***
***
a
***
***
***
***
***
**
***
***
*
***
a
**
***
a
***
*
*** *** ***
Acknowledgments I am sincerely grateful to Drs. Vasiliy Radashevsky and Alexey Chernishev for the given material, to Drs. Konstantin Lutaenko, and Jun Sang Lee for the identification of the studied oyster species. My special thanks to Dr. Evgeny Ivashkin for his help in the statistic analysis, to Natalia Miroshnikova and two anonymous reviewers for comments and editing of this manuscript. This work was supported by the grants of the Far East Branch of the Russian Academy of Sciences (# 09-III-A-06-215) and Russian Foundation for Basic Research (# 11-04-98555).
** a ***
References
***
Abbreviations: AL, acrosome length; AW, acrosome width; FL, anterior nuclear fossa length; FW, anterior nuclear fossa width; NL, nucleus length; NW, nucleus width. a No statistically significant difference. * P < 0.05. ** P < 0.01. *** P < 0.001.
to half of nuclear length (in C. gigas from Sea of Japan, S. cf. mordax) or be situated on the top of the nucleus forming small recess (in C. cf. rivularis). This parameter was statistically different between the studied species with various significance levels (Table 3). Unfortunately, the size characters were not taken into account in the earlier descriptions of sperm structure in ostreids (S. commercialis [Healy and Lester, 1991], C. virginica [Daniels et al., 1971; Eckelbarger and Davis, 1996], C. angulata and O. edulis [Sousa and Oliveira, 1994]); therefore, the comparative analysis is restricted to the group presented in this report. It may be recommended for the future sperm descriptions in ostreids to present both microscopic data and statistically calculated dimensional characters of the sperm components (acrosome, nucleus and anterior nuclear fossa) and size ratios between them. In the present study the spermatozoa of two C. gigas populations were carefully investigated. The morphometrical analysis demonstrates significant differences among almost all the ratios of sperm compartments (see Table 3); the ratio of width of the anterior nuclear fossa to width of the nucleus is the only feature that does not show any difference between C. gigas populations from the Sea of Japan and Taiwan Strait. Among other mollusk species interpopulation differences were recorded in Mytilus edulis from Kamchatka, the Sea of Japan and the White Sea (Drozdov and Reunov, 1986) and Mytilus galloprovincialis from U.K. and South Africa (Hodgson and Bernard, 1986). But the interpopulation differences in the acrosome and nucleus sizes were never more vivid than interspecific ones within the studied group (see Table 3). Therefore, the samples from Taiwan seem not to be a population of C. gigas, but a distinct species. Comparison between the linear sizes of nucleus and acrosome in C. gigas from Taiwan, C. gigas from the Sea of Japan, and C. angulata indicates that these individuals belong to C. angulata (Table 2). However, comprehensive studies including a number of
Asif, M., 1979. Hermaphroditism and sex reversal in the four common oviparous species of oysters from the coast of Karachi. Hydrobiologia 66, 49–55. Bozzo, M.G., Poquet, M., Sagristà, E., Durfort, M., 2008. Ultraestructura dels espermatozoides de Crassostrea gigas, Mytilus galloprovincialis and I Donax trunculus (Mollusca, Bivalvia). Treballs SCB 59, 59–70. Bozzo, M.G., Ribes, E., Sagristà, E., Poquet, M., Durfort, M., 1993. Fine structure of the spermatozoa of Crassostrea gigas (Mollusca, Bivalvia). Mol. Reprod. Dev. 34, 206–211. Brandriff, B., Moy, G.W., Vacquier, V.D., 1978. Isolation of sperm bindin from the oyster (Crassostrea gigas). Gamete Res. 1, 89–99. Buroker, N.E., 1985. Evolutionary patterns in the family Ostreidae: larviparity vs. oviparity. J. Exp. Mar. Biol. Ecol. 90, 233–247. Collado, G.A., Brown, D.I., 2006. Morphology of spermatozoon in two sympatric species of Fissurella Bruguière, 1789 (Mollusca: Vetigastropoda) from Southern Chile. Invertebr. Reprod. Dev. 49, 79–84. Collado, G.A., Espoz, C., Méndez, M.A., Brown, D.I., 2008. Spermatozoon ultrastructure in seven South American species of Tegula Lesson, 1835 (Mollusca: Vetigastropoda) and the phylogenetic implications for the subfamily Tegulinae. Invertebr. Reprod. Dev. 52, 13–21. Daniels, E.W., Longwell, A.C., Mcniff, J.M., 1971. Ultrastructure of spermatozoa from the American oyster Crassostrea virginica. Trans. Am. Microsc. Soc. 90, 275–282. Dong, Q., Huan, G.C., Tiersch, T.R., 2005. Spermatozoal ultrastructure of diploid and tetraploid Pacific oysters. Aquaculture 249, 487–496. Drozdov, A.L., Reunov, A.A., 1986. The morphology of gametes of the blue mussel Mytilus edulis from the White and Japan seas and Avacha inlet. Biol. Morya-Mar. Biol. 4, 52–55 (in Russian with English summary). Drozdov, A.L., Sharina, S.N., Tyurin, S.A., 2009. Sperm ultrastructure in representatives of six bivalve families from Peter the Great Bay, Sea of Japan. Russ. J. Mar. Biol. 35, 236–241. Eckelbarger, K.J., Davis, C.V., 1996. Ultrastructure of the gonad and gametogenesis in the eastern oyster, Crassostrea virginica. II. Testis and spermatogenesis. Mar. Biol. 127, 89–96. Eckelbarger, K.J., Young, C.M., 1999. Ultrastructure and gametogenesis in a chemosynthetic mytilid bivalve (Bathymodiolus childressi) from a bathyal, methane seep environment (northern Gulf of Mexico). Mar. Biol. 135, 635–646. Foighil, D.Ó., Taylor, D.J., 2000. Evolution of parental care and ovulation behavior in oysters. Mol. Phylogenet. Evol. 15, 301–313. Franzèn, A., 1983. Ultrastructural studies of spermatozoa in three bivalve species with notes on evolution of elongated sperm nucleus in primitive spermatozoa. Gamete Res. 7, 199–214. Gwo, J.C., Liou, C.H., Cheng, C.H., 1996. Ultrastructure of the spermatozoa of the pacific oyster Crassostrea gigas (Mollusca, Bivalvia, Ostreidae). J. Submicrosc. Cytol. Pathol. 28, 395–400. Healy, J.M., 1996. Molluscan sperm ultrastructure: correlation with taxonomic units within the Gastropoda, Cephalopoda and Bivalvia. In: Taylor, J. (Ed.), Origin and Evolutionary Radiation of the Mollusca. Oxford University Press, pp. 99–113. Healy, J.M., Beames, K.P., Barclay, D.B., 1998. Spermatozoa of the Australian greenlip abalone Haliotis laevigata Donovan: ultrastructure and comparison with other gastropods, especially other Haliotidae (Vetigastropoda, Mollusca). Invertebr. Reprod. Dev. 34, 197–206. Healy, J.M., Lester, R.J.G., 1991. Sperm ultrastructure in the Australian oyster Saccostrea commercialis (Iredale & Roughley) (Bivalvia: Osteroidea). J. Mollus. Stud. 57, 219–224.
O.V. Yurchenko / Micron 43 (2012) 365–373 Hodgson, A.N., Bernard, R.T.F., 1986. Observations on the ultrastructure of the spermatozoon of two mytilids from the south-west coast of England. JMBA 66, 385–390. Hodgson, A.N., Bernard, R.T.F., Van Der Horst, G., 1990. Comparative spermatology of three species of Donax (Bivalvia) from South Africa. J. Mollus. Stud. 56, 257–265. Hodgson, A.N., Chia, F.-S., 1993. Spermatozoon structure of some North-American prosobranchs from the families Lottiidae (Patellogastropoda) and Fissurellidae (Archaeogastropoda). Mar. Biol. 116, 97–101. Hodgson, A.N., De Villiers, C.J., Bernard, R.T.F., 1987. Comparative spermatology of two morphologically similar species of Solen (Mollusca: Bivalvia). S. Afr. J. Zool./S.-Afr. Tydskr. Dierkd. 22, 264–268. Introíni, G.O., De Magalhães, C.A., Aguiar Jr., O., Quaresma, A.J.C., Lino-Neto, J., ReccoPimentel, S.M., 2004. Spermatozoan morphology of Brachidontes darwinianus and Brachidontes solisianus (Bivalvia, Mytilidae) from the southern Brazilian coast. Invertebr. Reprod. Dev. 46, 149–158.
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Kafanov, A.I., Drozdov, A.L., 1998. Comparative sperm morphology and phylogenetic classification of recent Mytiloidea (Bivalvia). Malacologia 39, 129–139. Komaru, A., Konishi, K., Wada, K.T., 1994. Ultrastructure of spermatozoa from induced triploid Pacific oyster, Crassostrea gigas. Aquaculture 123, 217–222. Popham, J.D., 1979. Comparative spermatozoon morphology and bivalve phylogeny. Malacol. Rev. 12, 1–20. Sousa, M., Azevedo, C., 1988. Comparative silver staining analysis on spermatozoa of various invertebrate species. Int. J. Invert. Reprod. Dev. 13, 1–8. Sousa, M., Oliveira, E., 1994. An ultrastructural study of Crassostrea angulata (Mollusca, Bivalvia) spermatogenesis. Mar. Biol. 120, 545–551. Yang, W.-X., Zhu, J.-Q., 2011. Spermatozoal ultrastructure of ark clams of the superfamily Arcacea. In: Tiersch, T.R., Green, C.C. (Eds.), Cryopreservation in Aquatic Species. , 2nd ed. World Aquaculture Society, Baton Rouge, LA, USA, pp. 92–99. Yurchenko, O.V., Radashevsky, V.I., Reunov, A.A., 2010. Ultrastructural study of spermatogenesis in the Pacific oyster Crassostrea gigas (Bivalvia, Ostreidae) from the Sea of Japan. Invertebr. Zool. 7, 55–69.
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Comparative ultrastructural study of spermatozoa in some oyster species from the Asian-Pacific Coast Olga V. Yurchenko A.V. Zhirmunsky Institute of Marine Biology, Far Eastern Branch of Russian Academy of Science, Palchevsky Street, 17, Vladivostok 690041, Russia
a r t i c l e
i n f o
Article history: Received 16 August 2011 Received in revised form 26 September 2011 Accepted 26 September 2011 Keywords: Crassostrea cf. rivularis Crassostrea nippona Saccostrea cf. mordax Reproduction Sperm ultramorphology
a b s t r a c t Sperm organization in the oysters Crassostrea gigas, Crassostrea nippona, Crassostrea cf. rivularis and Saccostrea cf. mordax inhabiting Asian Pacific coast was studied. The spermatozoa of all studied species had a number of common morphological characters such as a cup-like acrosome with heterogeneous matrix on its top, an axial rod in the subacrosomal space, a barrel-shaped nucleus, four mitochondria in the midpiece, pericentriolar complexes, and a 9 + 2-organized flagellum. The spermatozoa of C. cf. rivularis differed from the other species by having cytoplasm processes in the midpiece region. Such structures have never been described in the Ostreidae. Additionally, each species could be identified by the shape and size of sperm compartments (acrosome, nucleus, anterior nuclear fossa). The most significant interspecific difference was found in the size of an anterior nuclear fossa. The smallest anterior nuclear fossa was found in C. cf. rivularis (about 0.24 m in length reaching about 22% of the nuclear length) while the biggest in C. gigas from the Sea of Japan (about 0.53 m in length reaching about 46% of the nuclear length). The spermatozoa of C. gigas collected from the Sea of Japan and Taiwan Strait differed significantly in almost all the studied parameters. Since sperm morphology has been successfully used for species differentiation, this suggests the existence of two species rather than two populations. The data obtained indicate the species-specific difference in the sperm ultrastructure within the Ostreidae, which may be identified both ultrastructurally and morphometrically. © 2011 Elsevier Ltd. All rights reserved.
1. Introduction Sperm morphology is believed to have useful application for taxonomic and phylogenetic analysis specifically at or above the family level (see Popham, 1979; Healy, 1996; Kafanov and Drozdov, 1998). But in Franzen’s (1983) opinion, there are not even two species of bivalve mollusk with identical spermatozoa. A number of studies have been conducted of closely related species. They show that even if the spermatozoa of these species are rather similar in their ultramorphology, they still can be recognized by morphometrical analysis or with cytochemical staining (genera Solen – Hodgson et al., 1987; Donax – Hodgson et al., 1990; Bathymodiolus – Eckelbarger and Young, 1999; Tegillarca – Yang and Zhu, 2011). Similar studies were conducted using mollusks of other taxa (genera Littorina – Sousa and Azevedo, 1988; Haliotis – see review by Healy et al., 1998; Brachidontes – Introíni et al., 2004; Fissurella – Collado and Brown, 2006; Tegula – Collado et al., 2008). To date the sperm structure has been studied in 5 oyster species among about 40 species of the family Ostreidae from all over the world: Saccostrea commercialis (Healy and Lester, 1991), Ostrea edulis (Sousa and Oliveira, 1994), Crassostrea virginica (Daniels et al.,
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1971; Eckelbarger and Davis, 1996), Crassostrea angulata (Sousa and Oliveira, 1994), Crassostrea gigas (Bozzo et al., 1993, 2008; Komaru et al., 1994; Gwo et al., 1996; Dong et al., 2005; Drozdov et al., 2009; Yurchenko et al., 2010). According to the available publications, there are two points of view on the species-specificity of oyster sperm. On the one hand, the spermatozoa of the studied species or even genera are very similar (for example, C. angulata and O. edulis) (Sousa and Oliveira, 1994). On the other hand the size and shape of the nucleus and acrosome may be species-specific (Gwo et al., 1996). To verify both points of view the present work was carried out. This paper studies the sperm ultrastructure and linear sizes of sperm head components in four species inhabiting the Asian Pacific coast (C. gigas, Crassostrea nippona, Crassostrea cf. rivularis, Saccostrea cf. mordax). Since our preliminary observations have indicated some dimensional difference in the sperm sizes between two populations of the pacific oyster C. gigas from Taiwan Strait and the Sea of Japan, the specimens from both populations were included into the list of studied oysters for detail investigation. 2. Materials and methods Oysters from four regions of the Asian Pacific coast were collected by SCUBA equipment. Some characters of individuals and
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Table 1 Characteristics of sampling sites and collected individuals. Oyster species
Sampling site
Date of sampling
Sample size (n)
Size of individuals (cm)
Crassostrea gigas (1)
Inner part of Vostok Bay, Peter the Great Bay of the Sea of Japan 42◦ 53 N 132◦ 44 E Korea Strait, Busan City coast (no precise coordinates) Tsaiyuan shallow water on Penghu Island, Penghu County in south-western Taiwan 23◦ 31 N 119◦ 33 E Nha Trang Bay, the South China Sea 12◦ 17 N 109◦ 14 E Nha Trang Bay, the South China Sea 12◦ 13 N 109◦ 13 E
June, 2006
5
10–15
August, 2009
3
20–22
May, 2006
5
10–15
March, 2009
5
7–9
June, 2010
3
4–5
Crassostrea nippona Crassostrea gigas (2)
Crassostrea cf. rivularis (Fig. 1A) Saccostrea cf. mordax (Fig. 1B)
sampling sites are shown in Table 1. The shell morphology of nondescript oyster species is presented in Fig. 1. The oysters were opened and their sex was identified after preparing gonad smears on object slides. Only males from each site were studied. Pieces of gonad tissues were fixed in 2.5% glutaraldehyde (Sigma) in 0.1 M cacodylate buffer (pH 7.5), with 21 mg/ml NaCl added, for 2 h at 4 ◦ C. The specimens were then rinsed several times in buffer, and transported to the laboratory of the Institute of Marine Biology (Vladivostok, Russia) at a temperature of +4 ◦ C. In the laboratory the material was post-fixed for 2 h with 2% osmium tetroxide prepared in the same buffer and then washed in buffer before dehydration. After dehydration in a graded ethanol series and acetone, the specimens were embedded in Spurr resin (Spurr,
EMS). The ultrathin sections (50–60 nm) were made using ultramicrotome Leica UC6 and stained with 2% aqueous uranyl acetate and lead citrate, and viewed with JEOL JEM 100S transmission electron microscope. To estimate the sizes of some sperm compartments (acrosome, nucleus and anterior nuclear fossa [Fig. 2]) 30 electronograms were made per species (10 for a male specimen, three males of each species) with spermatozoa in straight longitudinal projection (axial rod extended from basal part of nuclear fossa to acrosome). The width of acrosome was measured at the basal diameter and the width of the anterior nuclear fossa at the widest part. The results were expressed as the mean ± SD. The species were compared using Student‘s t-test at a significance level of 0.05.
Fig. 1. Oyster species from Nha Trang Bay (Vietnam). Left and right valves, external and internal views. (A) Crassostrea cf. rivularis and (B) Saccostrea cf. mordax.
O.V. Yurchenko / Micron 43 (2012) 365–373
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situated (Fig. 3 line C, C. cf. rivularis). They have also been recorded in transverse sections through the mitochondrial region (Fig. 3 line D, C. cf. rivularis). Flagellums have common 9 + 2 microtubule organization. 3.2. Dimensional characters
Fig. 2. Sperm organization in oysters. Scheme for measuring sperm compartments by electron microscopy.
3. Results 3.1. Ultramorphology The spermatozoa of all studied species consist of an acrosomal complex, a nucleus, a midpiece and a flagellum. Each nucleus is barrel-shaped, with granular electron dense chromatin; anterior and posterior fossae are located, respectively on the apical and basal parts of the nucleus (Figs. 3 line A and 4A). In all presented species the acrosomal complex includes an acrosome and a subacrosomal space full of flaky substance with moderate electron-density. The contents of the acrosome differentiate into a large and homogeneous basal region and a small heterogeneous apical region with laminar structure (Figs. 3 line B and 4B). The subacrosomal space is formed by the invaginated basal part of the acrosome and an anterior nuclear fossa and contains an axial rod extended from the basal part of the anterior nuclear fossa to the apical part of the subacrosomal space (Figs. 3 line B and 4B). The anterior nuclear fossa has different shapes in the studied species. In C. cf. rivularis it looks like a small depression, in S. cf. mordax it is deep and cylindrical (Fig. 4A), in C. gigas from the Japan Sea and Taiwan populations and in C. nippona cup-like (Fig. 3 line A). This visual difference is confirmed by morphometric analysis (see Section 3.2). Spermatozoa with an overacrosomal electron-lucent terminal knob were found in all studied species. The midpiece of spermatozoa consists of four mitochondria surrounding a centriolar complex (Figs. 3 line C and 4C). The proximal and distal centrioles orientated orthogonally to each other have accessory structures. The proximal centriole is connected to a short centriolar rootlet present as a conical mass and extended into the posterior nuclear fossa (Figs. 3 line D and 4D). The distal centriole has nine radially arranged satellite fibers attaching it to the plasma membrane (Figs. 3 line E and 4E). Among the studied species only C. cf. rivularis has a specific midpiece organization. In the mitochondrial region rather long cytoplasmic processes, about 1 m, are
Despite rather similar sperm ultramorphology, spermatozoa have species-specific linear dimensions of the nucleus, acrosome and anterior nuclear fossa (Table 2). For interspecific comparison, it seems reasonable to compare both linear sizes and linear ratios: length/width ratios of sperm compartments (for example, nucleus length to its width) and length ratios between sperm compartments (for example, acrosome length to nucleus length, etc.). Among the species the length/width ratio of the nuclei in the spermatozoa varies from 0.54 in C. gigas from Taiwan Strait to 0.69 in S. cf. mordax (Table 2); the difference was statistically significant both between these species and in comparison with other species (Fig. 5A and Table 3). The spermatozoa of Taiwan C. gigas are characterized by a shortened and wide acrosome (length/width ratio 0.43 vs 0.49–0.53 in the other species) (Fig. 5B). The ratio of acrosome length to nucleus length indicates that spermatozoa in S. cf. mordax and C. cf. rivularis have short acrosomes (Fig. 5C) constituting about 34–35% of nucleus length, while in C. gigas from both populations and C. nippona the acrosome is nearly half as long as the nucleus (Table 2). Only C. nippona and C. gigas from Taiwan Strait were almost similar in this parameter (Fig. 5C and Table 3). According to measuring, the spermatozoa of the studied species vary in the shapes and sizes of the anterior nuclear fossa (Table 2). S. cf. mordax is the only species with longer than wide anterior nuclear fossa (Table 2). In the other species the width was significantly bigger than the length (Table 2 and Fig. 5D). Although the linear sizes of C. nippona and C. cf. rivularis are notably unequal, the length/width ratios of the fossa do not show any significant difference (Table 3). The ratio of fossa length to nucleus length significantly varies among almost all the studied species (Fig. 5E) with the exception of S. cf. mordax, in which this parameter is similar to that of C. gigas from Taiwan Strait (Table 3). Interspecies difference in the ratio of anterior fossa width to nucleus width is less pronounced (Fig. 5F), and the anterior nuclear fossa occupies about 33–35% of the nucleus width excluding C. cf. rivularis and S. cf. mordax with rather compact nuclear fossae (23% and 24%, respectively). 4. Discussion 4.1. Ultrastructural characteristics The comparison of sperm structure in four species of Ostreidae shows that they closely resemble one another and those in other investigated ostreids (S. commercialis – Healy and Lester, 1991, O. edulis – Sousa and Oliveira, 1994, C. virginica – Daniels et al., 1971; Eckelbarger and Davis, 1996, C. angulata – Sousa and Oliveira, 1994, C. gigas – Bozzo et al., 1993; Komaru et al., 1994; Gwo et al., 1996; Dong et al., 2005; Drozdov et al., 2009; Yurchenko et al., 2010). However, the present study reveals some ultrastructural features that have not been described earlier. In the species studied here the electron-lucent terminal knob was often noticed in the tip of the acrosome beneath the cytoplasmic membrane. Since such a structure has earlier been found in C. gigas from the Sea of Japan but was not recorded in the C. gigas from other regions, it can be interpreted as different degrees of spermatozoon maturity rather than a “possible species-specific feature” (Yurchenko et al., 2010). In addition, the present wider study indicates that this knob can be found in other oyster species and cannot be accepted as an individual trait of C. gigas.
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Fig. 3. Sperm ultramorphology in the four oysters belonging to the genus Crassostrea. (See the species names on the top of the figure.) (Line A) Longitudinal section through the spermatozoon. (Line B) Longitudinal section through the acrosome and subacrosomal space (asterisk). (Line C) Transverse section through the midpiece. (Line D) Longitudinal section through the midpiece. (Line E) Section through the pericentriolar complex of the distal centriole. Abbreviations: a, acrosome; anf, anterior nuclear fossa; ar, axial rod; av, acrosomal vesicle; cp, cytoplasmic process; dc, distal centriole; f, flagellum; m, mitochondrion; n, nucleus; pc, proximal centriole; pnf, posterior nuclear fossa; r, rootlet; sf, satellite fibers; tk, terminal knob. Scale bars: (Line A) 1 m; (Line B) 0.3 m; (Lines C and D) 0.5 m and (Line E) 0.2 m.
Table 2 Summary of dimensions (m) of spermatozoa compartments in the family Ostreidae. Organelles
Crassostrea cf. rivularis Genus Saccostrea Saccostrea cf. mordax Saccostrea commercialis Genus Ostrea Ostrea edulis
a
Nucleus
Anterior nuclear fossa
Acrosome length/ nucleus length
Fossa length/ nucleus length
Fossa width/ nucleus width
Source
Length
Width
L/W
Length
Width
L/W
Length
Width
L/W
0.58 ± 0.05
1.09 ± 0.11
0.53 ± 0.05
1.15 ± 0.09
1.90 ± 0.19
0.61 ± 0.06
0.53 ± 0.08
0.62 ± 0.08
0.87 ± 0.15
0.50 ± 0.05
0.46 ± 0.07
0.33 ± 0.03
Present study
0.49 ± 0.05
1.13 ± 0.11
0.43 ± 0.03
1.07 ± 0.09
1.99 ± 0.19
0.54 ± 0.04
0.43 ± 0.06
0.67 ± 0.09
0.65 ± 0.11
0.46 ± 0.04
0.40 ± 0.05
0.33 ± 0.02
Present study
0.43 ± 0.06
0.85 ± 0.07
0.5
0.96 ± 0.08
1.55 ± 0.12
0.62
–
–
–
0.45
–
–
0.53 ± 0.07
1.02 ± 0.11
0.52 ± 0.05
1.12 ± 0.09
1.83 ± 0.17
0.61 ± 0.05
0.35 ± 0.04
0.64 ± 0.06
0.55 ± 0.09
0.47 ± 0.04
0.31 ± 0.05
0.35 ± 0.02
0.5
1.1
0.45
1.0
–
–
–
–
–
–
–
–
0.38 ± 0.05
0.74 ± 0.08
0.52 ± 0.04
1.09 ± 0.11
1.74 ± 0.16
0.62 ± 0.07
0.24 ± 0.04
0.41 ± 0.06
0.58 ± 0.09
0.35 ± 0.04
0.22 ± 0.04
0.24 ± 0.02
Gwo et al. (1996) Present study Sousa and Oliveira (1994) Present study
0.39 ± 0.05
0.8 ± 0.09
0.49 ± 0.04
1.17 ± 0.15
1.71 ± 0.19
0.69 ± 0.07
0.5 ± 0.09
0.39 ± 0.05
1.3 ± 0.22
0.34 ± 0.03
0.43 ± 0.05
0.23 ± 0.02
0.2
0.8–1.0
0.22
1.0
1.4–1.5
0.69
0.4–0.5
–
–
0.2
0.45
–
0.5
0.9
0.56
–
–
–
–
–
–
–
–
–
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Genus Crassostrea Crassostrea gigas (Sea of Japan) Crassostrea gigas (Taiwan Strait) Crassostrea gigas Crassostrea nippona Crassostrea angulata
Acrosomea
Present study Healy and Lester (1991) Sousa and Oliveira (1994)
The size includes the length of an electron-dense granule without an electron-lucent knob.
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An anterior banded substructure found both in the species described here and in the previously studied ones (aka “whorl-like component” [Brandriff et al., 1978] or “laminar-like portion of acrosomal vesicle” in C. gigas [Bozzo et al., 1993], “semicircular dense laminae” in C. angulata and O. edulis [Sousa and Oliveira, 1994] and “series of transverse bands” in S. commercialis [Healy and Lester, 1991]) seems to be an attribute of the family Ostreidea. It should be noted that in the previous reports the fixation procedure varied in the composition and concentration of primary fixers (from 2% to 5% glutaraldehyde or mixture of 2% paraformaldehyde and 2.5% glutaraldehyde prepared in cacodylate or phosphate buffers with the addition of NaCl or sucrose or van’t Hoff’s artificial sea water), as well as in the composition and concentration of staining solution of uranyl acetate (from 2% to 5% of aqueous or alcoholic solution); in some cases sections were treated with lead citrate only. Analysis of morphological difference in sperm ultrastructure made by Healy et al. (1998) showed that depending on fixation, cytochemical treatments and section staining methods employed, the same acrosomal components in the sperm of Haliotidae species may appear either more or less electron dense. Therefore, C. virginica, which has typical acrosome morphology but lacks the anterior banded substructure (Eckelbarger and Davis, 1996), is probably not an exception, and future reinvestigation using other fixers and subsequent treatment procedures may demonstrate that its acrosome has the similar fine organization. The presence of cytoplasmic processes in the midpiece region of spermatozoa is a species-specific trait of C. cf. rivularis. Among the mollusk species, the cytoplasmic structures in the midpiece of spermatozoa called “collar” or “membranous skirts” were noticed in some species of Patellogastropoda (see Hodgson and Chia, 1993) and Cephalopoda (see Healy, 1996). Their occurrence is likely to be connected with aspects of fertilization process, because spermatozoa with membranous skirt among Cephalopoda were attributed to species with fertilization within the mantle cavity (see Healy, 1996). The oysters of Ostrea and Lopha genera have a similar way of fertilization and are larviparous, while the oysters of Crassostrea and Saccostrea genera discharge their eggs and sperm directly into the surrounding seawater (Buroker, 1985; Foighil and Taylor, 2000). It is expectable, that Ostrea species could have sperm with accessory structures helping it to penetrate a female organism and attach and/or anchor to egg surface under stream conditions. But the organization of O. edulis spermatozoa does not show any accessories (Sousa and Oliveira, 1994). According to Asif (1979), C. rivularis is an oviparous species therefore the role of the cytoplasmic processes is related to some other aspect of reproductive biology and will have to be the subject of further discussion. 4.2. Dimensional characteristics In addition to morphological traits, the dimensional measurements of sperm components also demonstrate interspecies differences, which confirm the suggestion of Gwo et al. (1996) about the distinctions of linear sizes of acrosome and nucleus in ostreid species. Moreover, the acrosome/nucleus ratios underline the individuality of sperm organization in some species of the Ostreidae (Fig. 5). For example, C. gigas from Taiwan Strait differs from the others by a lower acrosome/nucleus length ratio, which makes the shape of sperm head more spherical. The spermatozoa of C. cf. rivularis and S. cf. mordax are distinguished among other studied species by a rather compact acrosome. In Table 2 the information
Fig. 4. Sperm ultramorphology of Saccostrea cf. mordax. (A) Longitudinal section through the spermatozoon. (B) Longitudinal section through the acrosome and subacrosomal space (asterisk). (C) Transverse section through the midpiece. (D) Longitudinal section through the midpiece. (E) Section through the pericentriolar
complex of the distal centriole. Abbreviations: a, acrosome; anf, anterior nuclear fossa; ar, axial rod; av, acrosomal vesicle; dc, distal centriole; m, mitochondrion; n, nucleus; pc, proximal centriole; pnf, posterior nuclear fossa; r, rootlet; sf, satellite fibers. Scale bars: (A) 1 m; (B) 0.3 m; (C and D) 0.5 m and (E) 0.2 m.
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Fig. 5. Whisker plots showing species-specific differences in the coefficients of sperm compartments ratios. Ratios of (A) nucleus length to nucleus width, (B) acrosome length to acrosome width, (C) acrosome length to nucleus length, (D) anterior nuclear fossa length to its width, (E) anterior nuclear fossa length to nucleus length and (F) anterior nuclear fossa width to nucleus width. X-axis shows: 1, Crassostrea gigas from the Sea of Japan; 2, C. nippona; 3, C. gigas from Taiwan Strait; 4, C. cf. rivularis and 5, Saccostrea cf. mordax.
on the size limits of the sperm compartments in the three genera of Ostreidae family is summarized. Since the dimensional data of species described in previous reports are rather scanty, it is difficult to single out some definite dimensional features that would be characteristic for the each genus; nevertheless, the suggestion
about different sizes of sperm compartments can be confirmed for the studied oysters inhabiting Asian coast. Along with nuclear and acrosomal sizes, the size of the anterior nuclear fossa may also be accepted as a vivid individual character in the studied species. Apparently, the nuclear fossa can occupy up
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Table 3 Significance values of pairwise test for the interspecific comparison of sperm compartments.
C. cf. rivularis
C. nippona
C. gigas (Taiwan Strait)
C. gigas (Sea of Japan)
Ratio
C. gigas (Sea of Japan)
NL/NW AL/NL AL/AW FL/NL FL/FW FW/NW NL/NW AL/NL AL/AW FL/NL FL/FW FW/NW NL/NW AL/NL AL/AW FL/NL FL/FW FW/NW NL/NW AL/NL AL/AW FL/NL FL/FW FW/NW
a
**
***
a
a
**
***
***
***
***
C. cf. rivularis
C. nippona
***
Saccostrea cf. mordax
morphological and molecular methods should be conducted to clarify its taxonomic position. Summing up the results, the ultrastructure and morphometrical analysis showed that each of the studied species had its unique sperm organization. Therefore, the mollusks from the family Ostreidae do not refute but confirm the Franzèn’s (1983) statement about presence of species-specific spermatozoa in bivalve mollusks. But whether described morphological and morphometrical features of spermatozoa involve into formation of interspecies reproductive barrier is required future experimental studies.
a
a
a
***
*
***
***
a
a
*
***
***
***
***
a
***
**
***
***
***
***
***
***
***
***
a
***
***
***
***
***
**
***
***
*
***
a
**
***
a
***
*
*** *** ***
Acknowledgments I am sincerely grateful to Drs. Vasiliy Radashevsky and Alexey Chernishev for the given material, to Drs. Konstantin Lutaenko, and Jun Sang Lee for the identification of the studied oyster species. My special thanks to Dr. Evgeny Ivashkin for his help in the statistic analysis, to Natalia Miroshnikova and two anonymous reviewers for comments and editing of this manuscript. This work was supported by the grants of the Far East Branch of the Russian Academy of Sciences (# 09-III-A-06-215) and Russian Foundation for Basic Research (# 11-04-98555).
** a ***
References
***
Abbreviations: AL, acrosome length; AW, acrosome width; FL, anterior nuclear fossa length; FW, anterior nuclear fossa width; NL, nucleus length; NW, nucleus width. a No statistically significant difference. * P < 0.05. ** P < 0.01. *** P < 0.001.
to half of nuclear length (in C. gigas from Sea of Japan, S. cf. mordax) or be situated on the top of the nucleus forming small recess (in C. cf. rivularis). This parameter was statistically different between the studied species with various significance levels (Table 3). Unfortunately, the size characters were not taken into account in the earlier descriptions of sperm structure in ostreids (S. commercialis [Healy and Lester, 1991], C. virginica [Daniels et al., 1971; Eckelbarger and Davis, 1996], C. angulata and O. edulis [Sousa and Oliveira, 1994]); therefore, the comparative analysis is restricted to the group presented in this report. It may be recommended for the future sperm descriptions in ostreids to present both microscopic data and statistically calculated dimensional characters of the sperm components (acrosome, nucleus and anterior nuclear fossa) and size ratios between them. In the present study the spermatozoa of two C. gigas populations were carefully investigated. The morphometrical analysis demonstrates significant differences among almost all the ratios of sperm compartments (see Table 3); the ratio of width of the anterior nuclear fossa to width of the nucleus is the only feature that does not show any difference between C. gigas populations from the Sea of Japan and Taiwan Strait. Among other mollusk species interpopulation differences were recorded in Mytilus edulis from Kamchatka, the Sea of Japan and the White Sea (Drozdov and Reunov, 1986) and Mytilus galloprovincialis from U.K. and South Africa (Hodgson and Bernard, 1986). But the interpopulation differences in the acrosome and nucleus sizes were never more vivid than interspecific ones within the studied group (see Table 3). Therefore, the samples from Taiwan seem not to be a population of C. gigas, but a distinct species. Comparison between the linear sizes of nucleus and acrosome in C. gigas from Taiwan, C. gigas from the Sea of Japan, and C. angulata indicates that these individuals belong to C. angulata (Table 2). However, comprehensive studies including a number of
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