- Email: [email protected]
Behavior of epixenosomes and the epixenosomal band during divisional morphogenesis in Euplotidium itoi (Ciliata, Hypotrichida)
Europ. J. Protisto!' 32, 77-80 (1996) February 23, 1996
European Journal of
PROTISTOLOGY
Behavior of Epixenosomes and the Epixenosomal Band During Divisional Morphogenesis in Euplotidium itoi (Ciliata, Hypotrichida) M. Anita Giambelluca and Giovanna Rosati Dipartimento di Scienze dell' Ambiente e del Territorio, Universita di Pisa, Pisa, Italy
SUMMARY In the present study the variations of the epixenosomal band and the behavior of epixenosomes during the divisional morphogenesis of Euplotodium itoi have been analyzed. From the results obtained it arises that the multiplication, the precise redistribution between the two daughter E. itoi cells, and the transformation of epixenosomes from form I to form II are all processes well coordinated with the reproductive cycle of the ciliate. Euplotidium can live and divide even without epixenosomes. It appears, however, that not only E. itoi has a strong influence on epixenosomes but that the epixenosomes themselves also influence the ciliate host in some way.
Introduction The hypotrich ciliate Euplotidium itoi harbors on its dorsal surface peculiar organisms referred to as epixenosomes [3, 4, 5]. As already described [5] epixenosomes can be found in two different forms: the reproductive form (form I) has a simple morphology, is able to divide and gradually transforms into the mature form (form II). Form II cannot undergo division and is equipped with a complex extrusive apparatus that carries genetic material at its tip when ejecting. For this reason we consider form II a possible propagative stage [5]. Epixenosomes are inserted in a cortical band located within well defined limits along both sides and at the anterior end of the E. itoi dorsal surface. In particular the left branch lies between the left margin of the ventral surface (where the marginal cirrus (Me) and the lateral adoral zone of membranelles (AZM) are inserted) and kinety I, the right branch lies between kineties IV and V, and the anterior "scarf" is delimited anteriorly by the collar AZM and posteriorly by the curved upper end of kinety I [5]. This cortical region has peculiar characteristics such as the presence of parasomal sac-like vesicles not associated with ciliary structures [1] and the ability to form deep depressions © 1996 by Gustav Fischer Verlag, Stuttgart
through which foreign bacteria and epixenosomes can be introduced inside the cytoplasm [2]. The significance of the relationship between the two organisms is still unknown. As at the end of E. itoi binary fission each daughter cell has a similar epixenosomal band. We analyzed the variations of the epixenosomal band and the behavior of epixenosomes during this process by means of scanning electron microscopy (SEM). From the data obtained, reported in this paper, it is evident that the multiplication, precise redistribution and transformation of epixenosomes from form I to form II are processes which are well coordinated with the reproductive cycle of E. itoi. Material and Methods Euplotidium itoi was collected from sea water along the shore located south of Leghorn (Italy) and cultured in the laboratory at 20-24 DC in filtered and pasteurized original sea water, periodically enriched with the green flagellate Dunalie/la salina and the diatom Pheodactilum tricornutum. For starvation experiments some specimens were isolated and maintained in sea water not enriched with food organisms up to 12 d. Some of these specimens were checked for the presence of epixenosomes after 6 and 12 d. The remaining 0932-4739-96-0032-0077$3.50-0
Fig. 1. The "scarf" of the epixenosomal band in the non dividing stage, x 1000. - Figs. 2-4. The scarf during morphogenesis.Fig. 2. An initial phase of the scarf widening. Note that the dorsal kineties are still unmodified, x 900. - Fig. 3. In this more advanced stage the scarf reaches its maximum width and shows a longitudinal furrow in the central region. The upper set of kineties bearing more numerous cilia can be seen, x 900. - Fig. 4. In correspondence to the furrow the majority ofepixenosomes are in form I, x 3500. - Fig. 5. Higher magnification of a part of the scarf: many form I epixenosomes are dividing (arrows indicate some of them), x 7000. - Figs. 6-8. The epixenosomal band in starved E. itoi. - Fig. 6. After 6 d starvation only a few epixenosomes form II are still present, x 1000. - Fig. 7. After 12 d starvation the cortex at the epixenosomal band level (arrow) is empty and smooth, x 1000. - Fig. 8. A morphogenetic stage of an E. itoi devoid of epixenosomes: its cortical region corresponding to the epixenosomal band does not widen, x 1000. K =Kineties; OP =oral primordium; I =form I epixenosomes; II =form II epixenosomes.
Epixenosomes During E. itoi Binary Fission . 79 organisms were then transferred to the enriched culture medium and allowed to begin their cell cycle again. Regularly fed Euplotidium were examined simultaneously as controls. For scanning electron microscopy (SEM) cultured or starved specimens were processed as described elsewhere [5].
Results The epixenosomal band during the non-dividing stage contains mainly form II epixenosomes and is 13-15 /lm in width (Fig. 1). Soon after the appearance of the macronuclear replication bands, i.e. during a very early stage of E. itoi morphogenesis, the epixenosomal band starts to widen (Fig. 2) and, although still lying within the same limits, reaches 20-25 J.!m in width before morphogenetical changes are visible along kineties in SEM preparations. At the same time the number of form I epixenosomes, mainly located in the central region of the band, increases. Then, roughly when the E. itoi macronucleus divides in two and the kineties appear separated in two distinct sets, in the central region of the band a deep, longitudinal furrow becomes evident (Fig. 3). This furrow is full of form I epixenosomes, easily recognizable (Fig. 4) because they are roundish and smaller than form II epixenosomes found along the peripheral, raised regions of the epixenosomal band. In Fig. 5, which shows a portion of the furrow at higher magnification, it is clearly visible that most form] epixenosomes are dividing. This situation persists until the accomplishment of E. itoi fission. With the lateral shifting of the forming opisthe, the epixenosomal band increases in length parallel with the developing opisthe's adoral zone of membranelles. Soon after separation both proter and opisthe still have a wide band. The central furrow, however, becomes rapidly less evident and as soon as the complete reabsorption of parental cirri occurs and the macronucleus divides into two pieces, the epixenosomal band returns to its typical width as during the non-dividing stage and contains more epixenosomes of form II than of form 1. The latter are still mainly found in the central region of the band.
Description of the Epixenosomal Band when E. itoi Division is Arrested When binary fission of E. itoi is prevented by starvation, epixenosomes are gradually lost. After a 6 - 7 day starvation only a few form II epixenosomes can be observed (Fig. 6); after l2 days the dorsal surface of E. itoi is empty and smooth (Fig. 7). When these starved Euplotidium are again given food most of them are able to recover and begin to divide again without epixenosomes. Apparently the cortical region corresponding to the epixenosomal band does not become wider during morphogenesis (Fig. 8).
Discussion The results reported here show that, also during morphogenesis, the cortical region corresponding to the epixenosomal band is a very specialized zone. Changes occur in this region according to a pattern which is well coordinated with morphogenetic events: the number of dividing epixenosomes begins to increase and the cortical region in which they lie begins to widen a little before the development of new ciliary structures becomes evident at the E. itoi cell surface. Then the multiplication of epixenosomes, the widening of the cortical band region, its increase in length due to lateral shifting, the appearance of the central furrow, and recovery of the typical non-dividing features, proceed along precise steps together with E. itoi morphogenesis. Interestingly, in specimens observed at TEM shortly after division, i.e. when the remaining old cirri are reabsorbed and the macronucleus divides into two pieces, most epixenosomes show a morphology half-way between form I and form II (Rosati, unpublished data). This explains why during the non-dividing stage form II epixenosomes are more abundant than form I. On the other hand, it is not clearly understood how, while the cortical region of the epixenosomal band narrows, the total number of epixenosomes decreases. It is possible that a spontaneous loss (detachment or ejection?) of old form II epixenosomes takes place: this is also suggested by the observation that during starvation Euplotidium have only form II epixenosomes and lose them gradually. The fact that the increase and decrease in the number of epixenosomes and the widening and narrowing of the cortical region in which they lie occur at the same time, reveals the close relationship existing between the two organisms, although it is evident that E. itoi can live and divide even without epixenosomes. In addition, when E. itoi stop dividing, so do epixenosomes. Moreover, when E. itoi devoid of epixenosomes undergo binary fission their cortical region corresponding to the epixenosomal band apparently does not widen. This not only indicates that the host cell strong influences the epixenosomes, but also that the epixenosomes themselves influence the ciliate at least to a certain extent.
References 1 Lenzi P. and Rosati G. (1993): Ultrastructural study of Euplotidium itoi (Ciliata Hypotrichida). Europ. J. Protista!.,
29,453-461.
2 Rosati G. (1994): Entry of foreign bacteria and its own epibionts (epixenosomes) in the ciliate Euplotidium itoi by cortical invaginations. Arch. Protistenk., 144, 283-288. 3 Rosati G., Verni E, and Lenzi P. (1993): "Epixenosomes": peculiar epibionrs of the ciliate Euplotidium itoi. The formation of the extrusive apparatus and the ejecting mechanism. Europ. J. Protista!., 29, 238-245.
80 . M. A. Giambelluca and G. Rosati 4 Rosati G., Lenzi P., and Verni F. (1993): "Epixenosomes": peculiar epibionts of the protozoon ciliate Euplotidium itoi: do their cytoplasmic tubules consist of tubulin? Micron, 24,465-471.
5 Verni F. and Rosati G. (1990): Peculiar epibionts in Euplotidium itoi (Ciliata, Hypotrichida). J. Protozool., 37,337343.
Key words: Epixenosomes - Symbiosis - Binary fission - Hypotrich ciliate - Euplotidium Giovanna Rosati, Dipartimento di Scienze dell' Ambiente e Territorio, Universita di Pisa, via A. Volta n. 4, 56126 Pisa, Italy
European Journal of
PROTISTOLOGY
Behavior of Epixenosomes and the Epixenosomal Band During Divisional Morphogenesis in Euplotidium itoi (Ciliata, Hypotrichida) M. Anita Giambelluca and Giovanna Rosati Dipartimento di Scienze dell' Ambiente e del Territorio, Universita di Pisa, Pisa, Italy
SUMMARY In the present study the variations of the epixenosomal band and the behavior of epixenosomes during the divisional morphogenesis of Euplotodium itoi have been analyzed. From the results obtained it arises that the multiplication, the precise redistribution between the two daughter E. itoi cells, and the transformation of epixenosomes from form I to form II are all processes well coordinated with the reproductive cycle of the ciliate. Euplotidium can live and divide even without epixenosomes. It appears, however, that not only E. itoi has a strong influence on epixenosomes but that the epixenosomes themselves also influence the ciliate host in some way.
Introduction The hypotrich ciliate Euplotidium itoi harbors on its dorsal surface peculiar organisms referred to as epixenosomes [3, 4, 5]. As already described [5] epixenosomes can be found in two different forms: the reproductive form (form I) has a simple morphology, is able to divide and gradually transforms into the mature form (form II). Form II cannot undergo division and is equipped with a complex extrusive apparatus that carries genetic material at its tip when ejecting. For this reason we consider form II a possible propagative stage [5]. Epixenosomes are inserted in a cortical band located within well defined limits along both sides and at the anterior end of the E. itoi dorsal surface. In particular the left branch lies between the left margin of the ventral surface (where the marginal cirrus (Me) and the lateral adoral zone of membranelles (AZM) are inserted) and kinety I, the right branch lies between kineties IV and V, and the anterior "scarf" is delimited anteriorly by the collar AZM and posteriorly by the curved upper end of kinety I [5]. This cortical region has peculiar characteristics such as the presence of parasomal sac-like vesicles not associated with ciliary structures [1] and the ability to form deep depressions © 1996 by Gustav Fischer Verlag, Stuttgart
through which foreign bacteria and epixenosomes can be introduced inside the cytoplasm [2]. The significance of the relationship between the two organisms is still unknown. As at the end of E. itoi binary fission each daughter cell has a similar epixenosomal band. We analyzed the variations of the epixenosomal band and the behavior of epixenosomes during this process by means of scanning electron microscopy (SEM). From the data obtained, reported in this paper, it is evident that the multiplication, precise redistribution and transformation of epixenosomes from form I to form II are processes which are well coordinated with the reproductive cycle of E. itoi. Material and Methods Euplotidium itoi was collected from sea water along the shore located south of Leghorn (Italy) and cultured in the laboratory at 20-24 DC in filtered and pasteurized original sea water, periodically enriched with the green flagellate Dunalie/la salina and the diatom Pheodactilum tricornutum. For starvation experiments some specimens were isolated and maintained in sea water not enriched with food organisms up to 12 d. Some of these specimens were checked for the presence of epixenosomes after 6 and 12 d. The remaining 0932-4739-96-0032-0077$3.50-0
Fig. 1. The "scarf" of the epixenosomal band in the non dividing stage, x 1000. - Figs. 2-4. The scarf during morphogenesis.Fig. 2. An initial phase of the scarf widening. Note that the dorsal kineties are still unmodified, x 900. - Fig. 3. In this more advanced stage the scarf reaches its maximum width and shows a longitudinal furrow in the central region. The upper set of kineties bearing more numerous cilia can be seen, x 900. - Fig. 4. In correspondence to the furrow the majority ofepixenosomes are in form I, x 3500. - Fig. 5. Higher magnification of a part of the scarf: many form I epixenosomes are dividing (arrows indicate some of them), x 7000. - Figs. 6-8. The epixenosomal band in starved E. itoi. - Fig. 6. After 6 d starvation only a few epixenosomes form II are still present, x 1000. - Fig. 7. After 12 d starvation the cortex at the epixenosomal band level (arrow) is empty and smooth, x 1000. - Fig. 8. A morphogenetic stage of an E. itoi devoid of epixenosomes: its cortical region corresponding to the epixenosomal band does not widen, x 1000. K =Kineties; OP =oral primordium; I =form I epixenosomes; II =form II epixenosomes.
Epixenosomes During E. itoi Binary Fission . 79 organisms were then transferred to the enriched culture medium and allowed to begin their cell cycle again. Regularly fed Euplotidium were examined simultaneously as controls. For scanning electron microscopy (SEM) cultured or starved specimens were processed as described elsewhere [5].
Results The epixenosomal band during the non-dividing stage contains mainly form II epixenosomes and is 13-15 /lm in width (Fig. 1). Soon after the appearance of the macronuclear replication bands, i.e. during a very early stage of E. itoi morphogenesis, the epixenosomal band starts to widen (Fig. 2) and, although still lying within the same limits, reaches 20-25 J.!m in width before morphogenetical changes are visible along kineties in SEM preparations. At the same time the number of form I epixenosomes, mainly located in the central region of the band, increases. Then, roughly when the E. itoi macronucleus divides in two and the kineties appear separated in two distinct sets, in the central region of the band a deep, longitudinal furrow becomes evident (Fig. 3). This furrow is full of form I epixenosomes, easily recognizable (Fig. 4) because they are roundish and smaller than form II epixenosomes found along the peripheral, raised regions of the epixenosomal band. In Fig. 5, which shows a portion of the furrow at higher magnification, it is clearly visible that most form] epixenosomes are dividing. This situation persists until the accomplishment of E. itoi fission. With the lateral shifting of the forming opisthe, the epixenosomal band increases in length parallel with the developing opisthe's adoral zone of membranelles. Soon after separation both proter and opisthe still have a wide band. The central furrow, however, becomes rapidly less evident and as soon as the complete reabsorption of parental cirri occurs and the macronucleus divides into two pieces, the epixenosomal band returns to its typical width as during the non-dividing stage and contains more epixenosomes of form II than of form 1. The latter are still mainly found in the central region of the band.
Description of the Epixenosomal Band when E. itoi Division is Arrested When binary fission of E. itoi is prevented by starvation, epixenosomes are gradually lost. After a 6 - 7 day starvation only a few form II epixenosomes can be observed (Fig. 6); after l2 days the dorsal surface of E. itoi is empty and smooth (Fig. 7). When these starved Euplotidium are again given food most of them are able to recover and begin to divide again without epixenosomes. Apparently the cortical region corresponding to the epixenosomal band does not become wider during morphogenesis (Fig. 8).
Discussion The results reported here show that, also during morphogenesis, the cortical region corresponding to the epixenosomal band is a very specialized zone. Changes occur in this region according to a pattern which is well coordinated with morphogenetic events: the number of dividing epixenosomes begins to increase and the cortical region in which they lie begins to widen a little before the development of new ciliary structures becomes evident at the E. itoi cell surface. Then the multiplication of epixenosomes, the widening of the cortical band region, its increase in length due to lateral shifting, the appearance of the central furrow, and recovery of the typical non-dividing features, proceed along precise steps together with E. itoi morphogenesis. Interestingly, in specimens observed at TEM shortly after division, i.e. when the remaining old cirri are reabsorbed and the macronucleus divides into two pieces, most epixenosomes show a morphology half-way between form I and form II (Rosati, unpublished data). This explains why during the non-dividing stage form II epixenosomes are more abundant than form I. On the other hand, it is not clearly understood how, while the cortical region of the epixenosomal band narrows, the total number of epixenosomes decreases. It is possible that a spontaneous loss (detachment or ejection?) of old form II epixenosomes takes place: this is also suggested by the observation that during starvation Euplotidium have only form II epixenosomes and lose them gradually. The fact that the increase and decrease in the number of epixenosomes and the widening and narrowing of the cortical region in which they lie occur at the same time, reveals the close relationship existing between the two organisms, although it is evident that E. itoi can live and divide even without epixenosomes. In addition, when E. itoi stop dividing, so do epixenosomes. Moreover, when E. itoi devoid of epixenosomes undergo binary fission their cortical region corresponding to the epixenosomal band apparently does not widen. This not only indicates that the host cell strong influences the epixenosomes, but also that the epixenosomes themselves influence the ciliate at least to a certain extent.
References 1 Lenzi P. and Rosati G. (1993): Ultrastructural study of Euplotidium itoi (Ciliata Hypotrichida). Europ. J. Protista!.,
29,453-461.
2 Rosati G. (1994): Entry of foreign bacteria and its own epibionts (epixenosomes) in the ciliate Euplotidium itoi by cortical invaginations. Arch. Protistenk., 144, 283-288. 3 Rosati G., Verni E, and Lenzi P. (1993): "Epixenosomes": peculiar epibionrs of the ciliate Euplotidium itoi. The formation of the extrusive apparatus and the ejecting mechanism. Europ. J. Protista!., 29, 238-245.
80 . M. A. Giambelluca and G. Rosati 4 Rosati G., Lenzi P., and Verni F. (1993): "Epixenosomes": peculiar epibionts of the protozoon ciliate Euplotidium itoi: do their cytoplasmic tubules consist of tubulin? Micron, 24,465-471.
5 Verni F. and Rosati G. (1990): Peculiar epibionts in Euplotidium itoi (Ciliata, Hypotrichida). J. Protozool., 37,337343.
Key words: Epixenosomes - Symbiosis - Binary fission - Hypotrich ciliate - Euplotidium Giovanna Rosati, Dipartimento di Scienze dell' Ambiente e Territorio, Universita di Pisa, via A. Volta n. 4, 56126 Pisa, Italy