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Computer-assisted reconstruction of the mating pairs of Aspidisca (Protozoa, Ciliophora)
Micron and MicroscopicaActa, Vol.20, No. 1, pp. 2~32, 1989. Printedin Great Britain.
0739~260/89 $3.00+0.00 © 1989MaxwellPergamonMacmillanplc
COMPUTER-ASSISTED RECONSTRUCTION OF THE MATING PAIRS OF ASPIDISCA (PROTOZOA, CILIOPHORA) GIOVANNA ROSATI,*:~ FRANCO VERNI* a n d PAOLO GUALTIERI t *Dipartimento Scienze dell'Ambiente e Territorio (Zoologia), Universit~ di Pisa, via A. Volta 4, 56100 Pisa, Italy tCNR--Instituto di Biofisica, via S. Lorenzo, 26-56100, Pisa, Italy (Received 10 March 1989; revised 4 April 1989)
Abstract--The ciliate Aspidisca shows a peculiar mating process during which the cytoplasmic bridge interconnecting the two mates, appears to vary in size and position. With the aid of a computer-assisted threedimensional reconstruction of newly formed and advanced pairs, it was shown that at the beginning of the mating process, the wails of the bridge were formed only by the ventral surfaces of both mates. It was also shown that later on, the dorsal surface of one partner is involved. This finding is considered as surprising, considering that in the ciliates studied to date, the fusion between the two mates always occurs at the level of a well defined region of the ventral surface, which is therefore considered as being a highly specialized zone. Index key words: Conjugation, computer-reconstruction, Aspidisca.
end of this rotation the two mates lie on nearly parallel planes, and the ventral surface of the upper one is in close contact with the dorsal surface of the other. During the rotation process the cytoplasmic bridge, which has connected the two mates from the beginning of the process, varies in size and position. In the present study, a computer-assisted, three-dimensional reconstruction of the bridge is newly formed and in advanced pairs of Aspidisca was performed to analyse the evolution of this fusion zone during the conjugative process.
INTRODUCTION In the life cycle of ciliates there is a sexual process in which cells of different mating types unite in pairs. They undergo meiosis and fertilization, and then reorganize themselves. The highly specialized hypotrichs commit the ventral cell surface, and more specifically, their peristomial fields in order to unite with each other during mating. Understandably, in this restricted area of the cell body, well determined events must occur for establishing cytoplasmic continuity between the two members of the pair. However, other similarly constructed genera have adopted rather different mating patterns to attain this goal as shown by the two hypotrichs (Euplotes and Oxytricha) studied to date in greater detail (Nobili, 1967; Ricci et al., 1975; Hammersmith, 1976; Dallai and Luporini, 1988). As recently reported by Dini et al. (1987), the mating pattern of Aspidisca differs from those of both the above mentioned genera. At the beginning of pair formation, the mates face each other, then they begin to turn around with respect to each other, always maintaining the correct homopolar antero-posterior orientation. At the
MATERIALS AND METHODS Two-day starved Aspidisca cells of different mating types were mixed together in order to obtain pairs. At two different time intervals, 1 h (young pairs) and 5 h (advanced pairs) from the onset of their formation, some pairs were isolated and fixed for TEM observation with a l:l mixture of 5 % glutaraldehyde in 0.2 M cacodylate buffer, pH 7.4, and 4% OsO 4 in distilled water. Following dehydration, the samples were then embedded in Epon Araldite mixture. Serial thin sections were cut using an LKB ultratome and stained with uranyl acetate and lead citrate.
:~To whom correspondence should be addressed. 27
28
G. Rosati, 1~. Vcrni and P. Gualtieri
A number of young and advanced pairs were processed for SEM observation following the technique described by Rosati et al. (1987). Three of the l-h-old pairs and three of the 5-h-old pairs were examined by means of a three-dimensional reconstruction technique. For each pair, up to 270 thin sections were obtained. These sections were collected on copper grids, 10 × 10: 26- 27 grids were obtained for each pair. Section thickness was estimated by light refraction (using gold-coloured sections) to be about 0.8 nm in thickness. One or two sections per grid were photographed and printed at a final magnificalion of ×2800. Each print was outlined on a transparent sheet and entered in the computer by means of a 12 × 12 digitizing tablet (Summagraphics) equipped with a four-button puck and connected to an IBM AT computer. Tracing could be followed on the computer monitor, where outlines were displayed as polygons. Alignment of successive sections was manually performed by the operator during the stacking procedure. The computer acquired the outlines by points according to a prefixed distance criterion (i.e. the greater the number of points, N), the smoother the profile. X, Y and Z coordinates were stored for each point, and the values for thickness and colours were stored for each outline. Different colours were used to identify each cell in the pair, and for each cell, its dorsal and ventral surfaces. Figures 3 and 6 show the top view of all the stacked sections of a young and of an advanced pair, respectively. This image was rotated, translated and scaled in the space by means of proper zones of the digitizing tablet, until a satisfactory viewpoint was obtained. These operations were performed by multiplying each polygon, which is stored in the computer as an N × 4 array (where N is the number of points of each outline, and the 4 refers to the X, Y, Z and T homogeneous coordinates), by the standard 4 x 4 transformation matrix (Rogers and Adams, 1976). When the viewpoint had been selected, in order to obtain the illusion of a three-dimensional effect, the surfaces of the mating cells were shaded by varying the brightness of different colours, using the Lambert's law as previously described by Baba et al. (1985). RESULTS AND DISCUSSION
The appearance of the young and advanced pairs as seen at the SEM is shown in Figs 1 and 2,
respectively. It is evident that the relative position of the mates and the position and characteristics of the fusion zone changed during the mating process. In young pairs the two mates are perpendicularly oriented to each other in such a way that the fusion zone appears partially hidden regardless of the viewpoint. In the case of the advanced pairs the two partners lie on nearly parallel planes and the bridge is clearly visible at least in the ventral view of the pairs. With the aid of the three-dimensional reconstruction, further information about the fusion zone was obtained. In Fig. 4 it appears that in young pairs the bridge is very thin ( ~ 3 lamt and involves exclusively the ventral surface of both partners. Moreover, no direct contact between the two mates is evident from the dorsal view (Fig. 5). The general configuration is quite different in the advanced pairs. Not only is the bridge thicker (5 ~tm), but also the involvement of the two partners in forming its delimiting surface is different. As shown in Fig. 7 from a ventral view, the bridge wall appears to be formed by the fusion of the ventral surfaces of both partners, although each is involved to a different extent. However, viewing the pair from a different point of view (Fig. 8), it can be clearly seen that the ventral surface of the upper partner directly fuses with the dorsal surface of the other one. Thus the fusion zone appears to involve both ventral and dorsal surfaces of one of the two mates (i.e. the mate which presumably maintains the original position (non-rotating partner, nR) while the other rotates (rotating partner, R) (Rosati et al., in preparation). We regard this finding as being quite new. Indeed, not only in hypotrichs as previously mentioned, but also in the ciliates studied to date in detail, the fusion between the two mates occurs only at the ventral surface, and, more specifically, at a particular zone (i.e. the peristomial area). The presence of a peristome with specialized ciliature differentiates the ventral from the dorsal surface in many ciliates. In hypotrichs, however, the difference is more evident, as these ciliates are generally dorso ventrally flattened with their dorsal surface convex and their ventral surface concave. The two surfaces appear to be different not only in morphology but also in function. The dorsal surface bearing only very short cilia (bristles) of probable sensorial meaning (Ruffolo, 1976; G6rtz, 1982), while the ventral surface bears specialized ciliature such as cirri and oral struc-
Reconstruction of Mating Pair of Aspidisca
Fig. 1. SEM of the ventral view of a young Aspidisca pair. The fusion zone (arrow) is partially hidden by the rotating partner (R) showing the dorsal surface. The non-rotating partner (nR) shows its ventral surface with the peristome (pe) and the locomotory ciliature (ci). Fig. 2. SEM of the ventral view of an advanced Aspidisca pair. The fusion zone (bracket) is clearly visible, Both partners show their ventral surface ( × 1000).
29
30
G. Rosati, F. Verni and P. Gualtieri
4
5 Figs 3-8. Computer generated pictures of Aspidisca pairs. The rotating partner has the ventral surface yellow and the dorsal surface red; the non-rotating partner has the ventral surface magenta and the dorsal surface green. Fig. 3: Top view of all stacked sections of a young pair. Fig. 4: A computer reconstructed picture of a young pair from a ventral view. The fusion zone is quite thin and involves the ventral surface of both partners. Fig. 5: Dorsal view of the same young pairs: no direct contact between the two mates is evident, Fig. 6: T o p view of all stacked sections of an advanced pair. Fig. 7: The ventral aspect of an advanced pair; the fusion zone involves the ventral surface of both partners. Fig. 8: A different view of the same advanced pair demonstrating that the fusion zone is wider and that a direct contact (arrow) between the ventral surface of R and the dorsal surface of nR is also occurring.
Reconstruction of Mating Pair of
i
Aspidisca
31
/'JI 6
7
32
G. Rosati, F. Verni and P. Gualtieri
tures. In some hypotrichs a difference between the two surfaces has been reported also at ultrastructural level (Nobili, 1967; Ruffolo, 1976; Wicklow, 1982; Rosati et al., 1987). It should be mentioned that on the plasma membrane the dorsal cell surface and the bristles of Aspidisca, there are numerous spherical bodies, measuring about 70 nm in diameter, which are lacking on the ventral side of the organism (Rosati et al., 1987). Although the artifact nature of these bodies cannot be completely excluded, their absence on the ventral surface is indicative of different behaviour of the two surfaces with respect to the same preparation techniques. This difference in surface behaviour can be explained only by admitting intrinsic differences. For these reasons, it is very interesting to note that the dorsal and the ventral surfaces of Aspidisca can fuse directly to each other, sharing such a specific and well determined function as the formation of the bridge
connecting the two mates during the conjugative process.
REFERENCES Baba, N., Naka, M., Muranaka, Y., Nakamura, S., Kino, 1. and Kanaya, K., 1985. Computer-aided stereographic
representatim: of an object reconstructed from micrographs of serial thin sections..~,lierotr .:vlicrosc. Acta, 15: 221 226. Dallai, R. and Luporini, P., 1989. Ultrastructural aspects of the development of the cytoplasmic connection between mating cells of the ciliate Euplotes crassus. Eur. Y. Pratistol., 24:125 132. Dini. F., Bracchi, P. and Gianni, A., 1987. Mating types m Aspidisea sp. (Ciliophora, Hypotrichida): a cluster of cryptic species. J. Protozool., 34:236 245. G6rtz, H. D., 1982. The behavior and line structure of the dorsal bristles of Euplotes mmuta, E. aediculatus and Stylonyehia mytilus (Ciliata, Hypotrichida ~. J. Protozool.. 29:353 359. Hammersmith, R. L., 1976. Differential cortical degradation in two members of early conjugant pairs of O\vtricha [allax. J. exp. Zool., 196:45 70. Nobili, R. 1967. Ultrastructure of the fusion region of conjugating Euplotes (Ciliata Hypotrichida)..'~,lonitore Zool. Ital. ( N S ) , !: 73 89. Ricci, N.. Banchetti, R., Nobili, R. and Esposito, t,. 1975. Conjugation in Oxytricha sp. (Hypotrichida, Ciliata} [. Morphocytological aspects. Aeta Protozool., 13:335 342. Rogers, D. F. and Adams, J. A., 1976. Mathematical Elements .lor Computer Graphics. McGraw-Hill, New York, pp. 46 86. Rosati, G., Verni, F., Bracchi, P. and Dini, F., 1987. An ultrastructural analysis of the ciliated protozoon 4spidisca sp. Trans. Am. Microse. Soe., 106:31 52. Ruffolo, J. J., 1976. Fine structure of the dorsal bristle complex and pellicle of Euplote~s. J. morphol., 148:469 488. Wicklow, B. J., 1983. Ultrastructure and cortical morphogenesis in the Euplotine Certesia quadrinucleata Fabredomergue, 1885 (Ciliophora, Protozoa). J. Protozool., 30: 256 266.
0739~260/89 $3.00+0.00 © 1989MaxwellPergamonMacmillanplc
COMPUTER-ASSISTED RECONSTRUCTION OF THE MATING PAIRS OF ASPIDISCA (PROTOZOA, CILIOPHORA) GIOVANNA ROSATI,*:~ FRANCO VERNI* a n d PAOLO GUALTIERI t *Dipartimento Scienze dell'Ambiente e Territorio (Zoologia), Universit~ di Pisa, via A. Volta 4, 56100 Pisa, Italy tCNR--Instituto di Biofisica, via S. Lorenzo, 26-56100, Pisa, Italy (Received 10 March 1989; revised 4 April 1989)
Abstract--The ciliate Aspidisca shows a peculiar mating process during which the cytoplasmic bridge interconnecting the two mates, appears to vary in size and position. With the aid of a computer-assisted threedimensional reconstruction of newly formed and advanced pairs, it was shown that at the beginning of the mating process, the wails of the bridge were formed only by the ventral surfaces of both mates. It was also shown that later on, the dorsal surface of one partner is involved. This finding is considered as surprising, considering that in the ciliates studied to date, the fusion between the two mates always occurs at the level of a well defined region of the ventral surface, which is therefore considered as being a highly specialized zone. Index key words: Conjugation, computer-reconstruction, Aspidisca.
end of this rotation the two mates lie on nearly parallel planes, and the ventral surface of the upper one is in close contact with the dorsal surface of the other. During the rotation process the cytoplasmic bridge, which has connected the two mates from the beginning of the process, varies in size and position. In the present study, a computer-assisted, three-dimensional reconstruction of the bridge is newly formed and in advanced pairs of Aspidisca was performed to analyse the evolution of this fusion zone during the conjugative process.
INTRODUCTION In the life cycle of ciliates there is a sexual process in which cells of different mating types unite in pairs. They undergo meiosis and fertilization, and then reorganize themselves. The highly specialized hypotrichs commit the ventral cell surface, and more specifically, their peristomial fields in order to unite with each other during mating. Understandably, in this restricted area of the cell body, well determined events must occur for establishing cytoplasmic continuity between the two members of the pair. However, other similarly constructed genera have adopted rather different mating patterns to attain this goal as shown by the two hypotrichs (Euplotes and Oxytricha) studied to date in greater detail (Nobili, 1967; Ricci et al., 1975; Hammersmith, 1976; Dallai and Luporini, 1988). As recently reported by Dini et al. (1987), the mating pattern of Aspidisca differs from those of both the above mentioned genera. At the beginning of pair formation, the mates face each other, then they begin to turn around with respect to each other, always maintaining the correct homopolar antero-posterior orientation. At the
MATERIALS AND METHODS Two-day starved Aspidisca cells of different mating types were mixed together in order to obtain pairs. At two different time intervals, 1 h (young pairs) and 5 h (advanced pairs) from the onset of their formation, some pairs were isolated and fixed for TEM observation with a l:l mixture of 5 % glutaraldehyde in 0.2 M cacodylate buffer, pH 7.4, and 4% OsO 4 in distilled water. Following dehydration, the samples were then embedded in Epon Araldite mixture. Serial thin sections were cut using an LKB ultratome and stained with uranyl acetate and lead citrate.
:~To whom correspondence should be addressed. 27
28
G. Rosati, 1~. Vcrni and P. Gualtieri
A number of young and advanced pairs were processed for SEM observation following the technique described by Rosati et al. (1987). Three of the l-h-old pairs and three of the 5-h-old pairs were examined by means of a three-dimensional reconstruction technique. For each pair, up to 270 thin sections were obtained. These sections were collected on copper grids, 10 × 10: 26- 27 grids were obtained for each pair. Section thickness was estimated by light refraction (using gold-coloured sections) to be about 0.8 nm in thickness. One or two sections per grid were photographed and printed at a final magnificalion of ×2800. Each print was outlined on a transparent sheet and entered in the computer by means of a 12 × 12 digitizing tablet (Summagraphics) equipped with a four-button puck and connected to an IBM AT computer. Tracing could be followed on the computer monitor, where outlines were displayed as polygons. Alignment of successive sections was manually performed by the operator during the stacking procedure. The computer acquired the outlines by points according to a prefixed distance criterion (i.e. the greater the number of points, N), the smoother the profile. X, Y and Z coordinates were stored for each point, and the values for thickness and colours were stored for each outline. Different colours were used to identify each cell in the pair, and for each cell, its dorsal and ventral surfaces. Figures 3 and 6 show the top view of all the stacked sections of a young and of an advanced pair, respectively. This image was rotated, translated and scaled in the space by means of proper zones of the digitizing tablet, until a satisfactory viewpoint was obtained. These operations were performed by multiplying each polygon, which is stored in the computer as an N × 4 array (where N is the number of points of each outline, and the 4 refers to the X, Y, Z and T homogeneous coordinates), by the standard 4 x 4 transformation matrix (Rogers and Adams, 1976). When the viewpoint had been selected, in order to obtain the illusion of a three-dimensional effect, the surfaces of the mating cells were shaded by varying the brightness of different colours, using the Lambert's law as previously described by Baba et al. (1985). RESULTS AND DISCUSSION
The appearance of the young and advanced pairs as seen at the SEM is shown in Figs 1 and 2,
respectively. It is evident that the relative position of the mates and the position and characteristics of the fusion zone changed during the mating process. In young pairs the two mates are perpendicularly oriented to each other in such a way that the fusion zone appears partially hidden regardless of the viewpoint. In the case of the advanced pairs the two partners lie on nearly parallel planes and the bridge is clearly visible at least in the ventral view of the pairs. With the aid of the three-dimensional reconstruction, further information about the fusion zone was obtained. In Fig. 4 it appears that in young pairs the bridge is very thin ( ~ 3 lamt and involves exclusively the ventral surface of both partners. Moreover, no direct contact between the two mates is evident from the dorsal view (Fig. 5). The general configuration is quite different in the advanced pairs. Not only is the bridge thicker (5 ~tm), but also the involvement of the two partners in forming its delimiting surface is different. As shown in Fig. 7 from a ventral view, the bridge wall appears to be formed by the fusion of the ventral surfaces of both partners, although each is involved to a different extent. However, viewing the pair from a different point of view (Fig. 8), it can be clearly seen that the ventral surface of the upper partner directly fuses with the dorsal surface of the other one. Thus the fusion zone appears to involve both ventral and dorsal surfaces of one of the two mates (i.e. the mate which presumably maintains the original position (non-rotating partner, nR) while the other rotates (rotating partner, R) (Rosati et al., in preparation). We regard this finding as being quite new. Indeed, not only in hypotrichs as previously mentioned, but also in the ciliates studied to date in detail, the fusion between the two mates occurs only at the ventral surface, and, more specifically, at a particular zone (i.e. the peristomial area). The presence of a peristome with specialized ciliature differentiates the ventral from the dorsal surface in many ciliates. In hypotrichs, however, the difference is more evident, as these ciliates are generally dorso ventrally flattened with their dorsal surface convex and their ventral surface concave. The two surfaces appear to be different not only in morphology but also in function. The dorsal surface bearing only very short cilia (bristles) of probable sensorial meaning (Ruffolo, 1976; G6rtz, 1982), while the ventral surface bears specialized ciliature such as cirri and oral struc-
Reconstruction of Mating Pair of Aspidisca
Fig. 1. SEM of the ventral view of a young Aspidisca pair. The fusion zone (arrow) is partially hidden by the rotating partner (R) showing the dorsal surface. The non-rotating partner (nR) shows its ventral surface with the peristome (pe) and the locomotory ciliature (ci). Fig. 2. SEM of the ventral view of an advanced Aspidisca pair. The fusion zone (bracket) is clearly visible, Both partners show their ventral surface ( × 1000).
29
30
G. Rosati, F. Verni and P. Gualtieri
4
5 Figs 3-8. Computer generated pictures of Aspidisca pairs. The rotating partner has the ventral surface yellow and the dorsal surface red; the non-rotating partner has the ventral surface magenta and the dorsal surface green. Fig. 3: Top view of all stacked sections of a young pair. Fig. 4: A computer reconstructed picture of a young pair from a ventral view. The fusion zone is quite thin and involves the ventral surface of both partners. Fig. 5: Dorsal view of the same young pairs: no direct contact between the two mates is evident, Fig. 6: T o p view of all stacked sections of an advanced pair. Fig. 7: The ventral aspect of an advanced pair; the fusion zone involves the ventral surface of both partners. Fig. 8: A different view of the same advanced pair demonstrating that the fusion zone is wider and that a direct contact (arrow) between the ventral surface of R and the dorsal surface of nR is also occurring.
Reconstruction of Mating Pair of
i
Aspidisca
31
/'JI 6
7
32
G. Rosati, F. Verni and P. Gualtieri
tures. In some hypotrichs a difference between the two surfaces has been reported also at ultrastructural level (Nobili, 1967; Ruffolo, 1976; Wicklow, 1982; Rosati et al., 1987). It should be mentioned that on the plasma membrane the dorsal cell surface and the bristles of Aspidisca, there are numerous spherical bodies, measuring about 70 nm in diameter, which are lacking on the ventral side of the organism (Rosati et al., 1987). Although the artifact nature of these bodies cannot be completely excluded, their absence on the ventral surface is indicative of different behaviour of the two surfaces with respect to the same preparation techniques. This difference in surface behaviour can be explained only by admitting intrinsic differences. For these reasons, it is very interesting to note that the dorsal and the ventral surfaces of Aspidisca can fuse directly to each other, sharing such a specific and well determined function as the formation of the bridge
connecting the two mates during the conjugative process.
REFERENCES Baba, N., Naka, M., Muranaka, Y., Nakamura, S., Kino, 1. and Kanaya, K., 1985. Computer-aided stereographic
representatim: of an object reconstructed from micrographs of serial thin sections..~,lierotr .:vlicrosc. Acta, 15: 221 226. Dallai, R. and Luporini, P., 1989. Ultrastructural aspects of the development of the cytoplasmic connection between mating cells of the ciliate Euplotes crassus. Eur. Y. Pratistol., 24:125 132. Dini. F., Bracchi, P. and Gianni, A., 1987. Mating types m Aspidisea sp. (Ciliophora, Hypotrichida): a cluster of cryptic species. J. Protozool., 34:236 245. G6rtz, H. D., 1982. The behavior and line structure of the dorsal bristles of Euplotes mmuta, E. aediculatus and Stylonyehia mytilus (Ciliata, Hypotrichida ~. J. Protozool.. 29:353 359. Hammersmith, R. L., 1976. Differential cortical degradation in two members of early conjugant pairs of O\vtricha [allax. J. exp. Zool., 196:45 70. Nobili, R. 1967. Ultrastructure of the fusion region of conjugating Euplotes (Ciliata Hypotrichida)..'~,lonitore Zool. Ital. ( N S ) , !: 73 89. Ricci, N.. Banchetti, R., Nobili, R. and Esposito, t,. 1975. Conjugation in Oxytricha sp. (Hypotrichida, Ciliata} [. Morphocytological aspects. Aeta Protozool., 13:335 342. Rogers, D. F. and Adams, J. A., 1976. Mathematical Elements .lor Computer Graphics. McGraw-Hill, New York, pp. 46 86. Rosati, G., Verni, F., Bracchi, P. and Dini, F., 1987. An ultrastructural analysis of the ciliated protozoon 4spidisca sp. Trans. Am. Microse. Soe., 106:31 52. Ruffolo, J. J., 1976. Fine structure of the dorsal bristle complex and pellicle of Euplote~s. J. morphol., 148:469 488. Wicklow, B. J., 1983. Ultrastructure and cortical morphogenesis in the Euplotine Certesia quadrinucleata Fabredomergue, 1885 (Ciliophora, Protozoa). J. Protozool., 30: 256 266.