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Mastigonemes in a bodonid flagellate
Experimental
MASTIGONEMES
Cell Research
IN A BODONID
37, 300-305
(1965)
FLAGELLATE
B. E. BROOKER Department
of Zoology,
University
Received
College
March
London,
England
16. 1964
MASTIGONEMES are lateral,
filamentous appendages of flagella. They have long been known to be present on the flagella of a number of protista, spermaof some fungi and tozoids and zoospores of some algae, and zoospores recently, have been reported from the choanocyte flagella of sponges [ 1 j. They may be present on one or both sides of the flagellum, conditions referred to by Deflandre [3] as stichonematic and pantonematic respectively. Each one of these conditions tends to be restricted to particular taxonomic groups [12]. Thus the stichonematic condition is found in the locomotory flagellum of euglenoids and the transverse flagellum of some dinoflagellates, the pantonematic flagellum in the chrysomonads, xanthomonads and brown algae [Y]. The present work describes a flagellum of the stichonematic type in a bodonid flagellate; mastigonemes have not previously been recorded from this group.
MATERIALS
AND
METHODS
A biflagellate protomonad from brackish water was recognised as a speciesof identified as Bodo sultans. It was isolated and maintained in a cloned culture in 1 per cent “Oxoid” liver infusion. Flagellates were examined with the light microscope using dark-field illumination, phase contrast and the flagella staining techniques of Deflandre [2] and Loeffler [4]. Pellets of the flagellates were prepared by gentle centrifugation of the culture fluid and then fixed at 4°C either in 1 per cent osmium tetroxide buffered with Verona1 acetate or in a IO per cent solution of formalin in M/15 Siirensenphosphate buffer; Bodo and tentatively
a./., anterior flagellum; CIX., axoneme; lum; f.m., flagellar membrane; m.b., flagellum. Fig. l.-Transverse x 54,000.
section
b., body of flagellate; bact., bacterium; mastigoneme bundles; pa.r., paraxial
of a flagellum
to show
the axoneme
(“9 + 2” fibrils)
b.f., bacterial flagclrod; p.f., posterior and paraxial
rod.
Fig. Z.-Whole Bode sultans to show the presence of mastigonemes on the anterior flagellum and their absence from the posterior flagellum. Note the regular spacing of the mastigoneme bundles and their termination at a point corresponding to that of the paraxial rod (arrowed). Segatively stained. x 14,200. Fig. S.-Part lar membrane.
of anterior Negatively
flagellum stained.
Experimenlal
Cell Research
37
suggesting x 28,000.
that
the mastigoneme
bundles
penetrate
the flagel-
Mastigonemes
in a bodonid flagellate
Bxperin~erdul
301
(:e/l
I~e.wnrcR
37
B. E. Brooker
302
some flagellates were further treated in an unfixed condition. A range of pH values (6.2-9.0) was used in fixation; the pH of the harvested cultures was 7.4. The pellets were then resuspended in the appropriate buffer and either negatively stained using a 1 per cent solution of phosphotungstic acid neutralised with sodium hydroxide or mounted directly on grids and shadowed with palladium. Although most observations were carried out on whole animals, some material was examined after embedding and sectioning. Pellets of the flagellates were prepared as above and fixed at 4°C in 1 per cent osmium tetroxide buffered to pH 7.4 with Verona1 acetate. This was followed by dehydration in a graded series of ethanolwater mixtures and then staining in a 1 per cent solution of phosphotungstic acid in absolute ethyl alcohol for I hr. The pellets were embedded in Araldite and sections cut on a Porter-Blum microtome. All electron microscopy was done using a Siemens Elmiskop 1 at 80 kV. RESULTS
AND
DISCUSSION
It was found that osmium tetroxide fixation produced the best preservation of the structures described below and that formalin or dry fixation was poor in this respect. Though mastigonemes were equally well demonstrated by metal shadowing and negatively staining, the latter gave by far the greatest detail of structure. Most of the results below were obtained using this technique. Bodo salfans has two flagella. One is anteriorly directed and serves to propel the animal; the second flagellum is longer and trails behind the animal when it is moving. In transverse section both contain the usual “9 + 2” pattern of fibrils (axoneme) and a paraxial rod (Fig. 1). Previous workers have described Bodo as having flagella of the acronematic type, i.e. bearing a single terminal filament [lo, 121, but have not reported structures resembling mastigonemes. The present work has shown that, in B. salfans at least, these structures are present, but only on the anterior flagellum (Fig. 2). The mastigonemes are arranged on the flagellum in a single row and so, using the terminology of Deflandre [3], the anterior flagellum can be classified as stichonematic: more correctly, since this flagellum is also acronematic, the term stichacronematic should be applied. This appears to be the first report of such a flagellum. Fig. 4.-Part of anterior flagellum to show nemes. Negatively stained. x 25,000.
that
each bundle
is made
up of a number
of mastigo-
Fig. 5.-An entire anterior flagellum. The mastigoneme bundles arise from the flagellum out of the plane of the paper and have been folded over to one side. Note that they form a single row. Metal shadowed with palladium. x 10,000. Fig. 6.-Longitudinal x 61,000. Experimenfal
section
Cell Research
37
through
the
tip
of a flagellum
to show
mastigoneme
bundles.
Mastigonemes
in a bodonid
flagellate
Experimental
303
Cell Research
37
301
B. E. Brooker
Unlike other protozoan mastigonemes, those of Bodo saltans arise from the flagellum in bundles (Fig. 4). It seems unlikely that this effect is due to irregular clumping of evenly-distributed mastigonemes, since the bundles show a regular periodicity of about 0.6 ,u and no free mastignonemes are found between them. Mastigoneme bundles have been seen in sectioned material (Fig. S), so it is improbable that they are artifacts produced by the drying of the flagella on the grids. The bundles taper from base to tip, and vary in number according to the state of preservation; the largest number seen on one flagellum is ten. The number of individual mastigonemes in each bundle also appears to vary, but well preserved specimens show 6-7 per bundle. The bundles have a base diameter of about 0.2 p and in length range from 1.2 ,u to 1.8 ,u; in any one individual their length tends to be constant. Pitelka and Schooley [lo] have reported that the appearance of the mastigonemes of Euglena can be changed by appropriately altering the pH of the fixative: this was not found to be so for the mastigonemes of Bodo saltans over the range of pH values used here. The mastigoneme bundles on whole mounts are found at regular intervals starting at a point about one quarter of the way up the flagellum and finishing at a point which coincides with the termination of the paraxial rod (Figs. 2, 5 and 6). They have never been seen beyond this point and arc always found on the same side of the flagellum as the paraxial rod. The possibility exists, therefore, that there is some relationship between the two structures. Since some flagellates with mastigoneme-bearing flagella do not contain a paraxial rod, this association is unlikely to be a fundamental one. In this case, it may simply be that the mastigonemes arise from that side of the flagellum which contains the paraxial rod. The point of origin of mastigonemes on flagella is as yet uncertain. Manton [6, 71 concludes from her work that they arise from the axoneme, whereas Pitelka and Schooley [lo] indicate that in Euglena and Ochromonas they appear to arise from the sheath. In Bodo saltans, several electron micrographs suggest that the mastigonemes pass through the flagellar membrane and therefore probably originate from either the paraxial rod or the axoneme (Fig. 3). It has been suggested that mastigonemes serve to increase the surface area of the flagellum, a factor which would result in an increased mechanical effectiveness of each flagellar beat [<5, 111. This theory is supported by the fact that in biflagellate cells, only one flagellum of which bears mastigonemes, it is usually the more active flagellum which has them. In Bodo saltans, although both flagella are capable of movement, the main propulsive force is generated Experimental
Cell
Research
37
Mastigonemes by the anterior flagellum. lends support to the above Light microscope studies fact that both anterior and have not yet been observed
303
in a bodonid flagellate
The fact that this is the one bearing mastigonemes, suggestion. have as yet led only to the confirmation of the posterior flagella are acronematic. Mastigonemes in either living or fixed material. SUMMARY
1. Mastigonemes are described from the anterior flagellum of a species of Bodo for the first time. 2. The mastigonemes are arranged in regularly spaced bundles in a single row, i.e. stichonematic. As the flagella are also known to be acronematic, the term stichacronematic may be used to describe this type of flagellum. 3. The mastigoneme bundles have been observed with the electron microscope on unfixed and on formalin and osmium tetroxide fixed, dried flagella. They have not been seen with the light microscope. 4. The origin and function of these structures is discussed. I wish to thank Dr D. R. Pitelka, Dr G. F. Leedale and Dr K. Vickerman for their advice and Dr E. G. Gray for the use of his electron microscope laboratory. Also, Mr K. Watkins for valuable technical assistance rendered. This work was carried out during the tenure of a postgraduate studentship from the Department of Scientific and Industrial Research. REFERENCES
Nature 191, 1319 (1961). G., Bull. Sot. Bofan. France 70, 738 (1923). __ Ann. Profist., Paris 4, 31 (1934). LOEFFLER, F., Zentr. Bakt. 6, 209 (1889). MAINX, F., Arch. Protisfenk. 60, 305 (1928). MANTON, I., Proc. Leeds Phil. Lit. Sot. Sci. Sec. 6, 306 (1955). __ J. Expfl Bof. 10, 448 (1959). PITELKA, D. R., Uniu. Calif. Pubf. Zodl. 53, 377 (1949).
1. AFZELIUS, 2. DEFLANDRE,
3. 4.
5. 6. 7. 8. 9. 10. 11. 12.
B. A.,
__ Electron-Microscopic Structure of Protozoa. Per&non Press, Oxford, PITELKA, D. R. and SCHOOLEY, C. N., Uniu. Calif. Publ. ZoBf. 61, 79 (1955). PRINGSHEIM, E. G. and HOVASSE, R., Arch. Zool. Expff. Gen. 86, 499 (1950). VLK, W., Arch. Profisfenk. 90, 448 (1938).
Experimenfal
1963.
Cell Research
37
MASTIGONEMES
Cell Research
IN A BODONID
37, 300-305
(1965)
FLAGELLATE
B. E. BROOKER Department
of Zoology,
University
Received
College
March
London,
England
16. 1964
MASTIGONEMES are lateral,
filamentous appendages of flagella. They have long been known to be present on the flagella of a number of protista, spermaof some fungi and tozoids and zoospores of some algae, and zoospores recently, have been reported from the choanocyte flagella of sponges [ 1 j. They may be present on one or both sides of the flagellum, conditions referred to by Deflandre [3] as stichonematic and pantonematic respectively. Each one of these conditions tends to be restricted to particular taxonomic groups [12]. Thus the stichonematic condition is found in the locomotory flagellum of euglenoids and the transverse flagellum of some dinoflagellates, the pantonematic flagellum in the chrysomonads, xanthomonads and brown algae [Y]. The present work describes a flagellum of the stichonematic type in a bodonid flagellate; mastigonemes have not previously been recorded from this group.
MATERIALS
AND
METHODS
A biflagellate protomonad from brackish water was recognised as a speciesof identified as Bodo sultans. It was isolated and maintained in a cloned culture in 1 per cent “Oxoid” liver infusion. Flagellates were examined with the light microscope using dark-field illumination, phase contrast and the flagella staining techniques of Deflandre [2] and Loeffler [4]. Pellets of the flagellates were prepared by gentle centrifugation of the culture fluid and then fixed at 4°C either in 1 per cent osmium tetroxide buffered with Verona1 acetate or in a IO per cent solution of formalin in M/15 Siirensenphosphate buffer; Bodo and tentatively
a./., anterior flagellum; CIX., axoneme; lum; f.m., flagellar membrane; m.b., flagellum. Fig. l.-Transverse x 54,000.
section
b., body of flagellate; bact., bacterium; mastigoneme bundles; pa.r., paraxial
of a flagellum
to show
the axoneme
(“9 + 2” fibrils)
b.f., bacterial flagclrod; p.f., posterior and paraxial
rod.
Fig. Z.-Whole Bode sultans to show the presence of mastigonemes on the anterior flagellum and their absence from the posterior flagellum. Note the regular spacing of the mastigoneme bundles and their termination at a point corresponding to that of the paraxial rod (arrowed). Segatively stained. x 14,200. Fig. S.-Part lar membrane.
of anterior Negatively
flagellum stained.
Experimenlal
Cell Research
37
suggesting x 28,000.
that
the mastigoneme
bundles
penetrate
the flagel-
Mastigonemes
in a bodonid flagellate
Bxperin~erdul
301
(:e/l
I~e.wnrcR
37
B. E. Brooker
302
some flagellates were further treated in an unfixed condition. A range of pH values (6.2-9.0) was used in fixation; the pH of the harvested cultures was 7.4. The pellets were then resuspended in the appropriate buffer and either negatively stained using a 1 per cent solution of phosphotungstic acid neutralised with sodium hydroxide or mounted directly on grids and shadowed with palladium. Although most observations were carried out on whole animals, some material was examined after embedding and sectioning. Pellets of the flagellates were prepared as above and fixed at 4°C in 1 per cent osmium tetroxide buffered to pH 7.4 with Verona1 acetate. This was followed by dehydration in a graded series of ethanolwater mixtures and then staining in a 1 per cent solution of phosphotungstic acid in absolute ethyl alcohol for I hr. The pellets were embedded in Araldite and sections cut on a Porter-Blum microtome. All electron microscopy was done using a Siemens Elmiskop 1 at 80 kV. RESULTS
AND
DISCUSSION
It was found that osmium tetroxide fixation produced the best preservation of the structures described below and that formalin or dry fixation was poor in this respect. Though mastigonemes were equally well demonstrated by metal shadowing and negatively staining, the latter gave by far the greatest detail of structure. Most of the results below were obtained using this technique. Bodo salfans has two flagella. One is anteriorly directed and serves to propel the animal; the second flagellum is longer and trails behind the animal when it is moving. In transverse section both contain the usual “9 + 2” pattern of fibrils (axoneme) and a paraxial rod (Fig. 1). Previous workers have described Bodo as having flagella of the acronematic type, i.e. bearing a single terminal filament [lo, 121, but have not reported structures resembling mastigonemes. The present work has shown that, in B. salfans at least, these structures are present, but only on the anterior flagellum (Fig. 2). The mastigonemes are arranged on the flagellum in a single row and so, using the terminology of Deflandre [3], the anterior flagellum can be classified as stichonematic: more correctly, since this flagellum is also acronematic, the term stichacronematic should be applied. This appears to be the first report of such a flagellum. Fig. 4.-Part of anterior flagellum to show nemes. Negatively stained. x 25,000.
that
each bundle
is made
up of a number
of mastigo-
Fig. 5.-An entire anterior flagellum. The mastigoneme bundles arise from the flagellum out of the plane of the paper and have been folded over to one side. Note that they form a single row. Metal shadowed with palladium. x 10,000. Fig. 6.-Longitudinal x 61,000. Experimenfal
section
Cell Research
37
through
the
tip
of a flagellum
to show
mastigoneme
bundles.
Mastigonemes
in a bodonid
flagellate
Experimental
303
Cell Research
37
301
B. E. Brooker
Unlike other protozoan mastigonemes, those of Bodo saltans arise from the flagellum in bundles (Fig. 4). It seems unlikely that this effect is due to irregular clumping of evenly-distributed mastigonemes, since the bundles show a regular periodicity of about 0.6 ,u and no free mastignonemes are found between them. Mastigoneme bundles have been seen in sectioned material (Fig. S), so it is improbable that they are artifacts produced by the drying of the flagella on the grids. The bundles taper from base to tip, and vary in number according to the state of preservation; the largest number seen on one flagellum is ten. The number of individual mastigonemes in each bundle also appears to vary, but well preserved specimens show 6-7 per bundle. The bundles have a base diameter of about 0.2 p and in length range from 1.2 ,u to 1.8 ,u; in any one individual their length tends to be constant. Pitelka and Schooley [lo] have reported that the appearance of the mastigonemes of Euglena can be changed by appropriately altering the pH of the fixative: this was not found to be so for the mastigonemes of Bodo saltans over the range of pH values used here. The mastigoneme bundles on whole mounts are found at regular intervals starting at a point about one quarter of the way up the flagellum and finishing at a point which coincides with the termination of the paraxial rod (Figs. 2, 5 and 6). They have never been seen beyond this point and arc always found on the same side of the flagellum as the paraxial rod. The possibility exists, therefore, that there is some relationship between the two structures. Since some flagellates with mastigoneme-bearing flagella do not contain a paraxial rod, this association is unlikely to be a fundamental one. In this case, it may simply be that the mastigonemes arise from that side of the flagellum which contains the paraxial rod. The point of origin of mastigonemes on flagella is as yet uncertain. Manton [6, 71 concludes from her work that they arise from the axoneme, whereas Pitelka and Schooley [lo] indicate that in Euglena and Ochromonas they appear to arise from the sheath. In Bodo saltans, several electron micrographs suggest that the mastigonemes pass through the flagellar membrane and therefore probably originate from either the paraxial rod or the axoneme (Fig. 3). It has been suggested that mastigonemes serve to increase the surface area of the flagellum, a factor which would result in an increased mechanical effectiveness of each flagellar beat [<5, 111. This theory is supported by the fact that in biflagellate cells, only one flagellum of which bears mastigonemes, it is usually the more active flagellum which has them. In Bodo saltans, although both flagella are capable of movement, the main propulsive force is generated Experimental
Cell
Research
37
Mastigonemes by the anterior flagellum. lends support to the above Light microscope studies fact that both anterior and have not yet been observed
303
in a bodonid flagellate
The fact that this is the one bearing mastigonemes, suggestion. have as yet led only to the confirmation of the posterior flagella are acronematic. Mastigonemes in either living or fixed material. SUMMARY
1. Mastigonemes are described from the anterior flagellum of a species of Bodo for the first time. 2. The mastigonemes are arranged in regularly spaced bundles in a single row, i.e. stichonematic. As the flagella are also known to be acronematic, the term stichacronematic may be used to describe this type of flagellum. 3. The mastigoneme bundles have been observed with the electron microscope on unfixed and on formalin and osmium tetroxide fixed, dried flagella. They have not been seen with the light microscope. 4. The origin and function of these structures is discussed. I wish to thank Dr D. R. Pitelka, Dr G. F. Leedale and Dr K. Vickerman for their advice and Dr E. G. Gray for the use of his electron microscope laboratory. Also, Mr K. Watkins for valuable technical assistance rendered. This work was carried out during the tenure of a postgraduate studentship from the Department of Scientific and Industrial Research. REFERENCES
Nature 191, 1319 (1961). G., Bull. Sot. Bofan. France 70, 738 (1923). __ Ann. Profist., Paris 4, 31 (1934). LOEFFLER, F., Zentr. Bakt. 6, 209 (1889). MAINX, F., Arch. Protisfenk. 60, 305 (1928). MANTON, I., Proc. Leeds Phil. Lit. Sot. Sci. Sec. 6, 306 (1955). __ J. Expfl Bof. 10, 448 (1959). PITELKA, D. R., Uniu. Calif. Pubf. Zodl. 53, 377 (1949).
1. AFZELIUS, 2. DEFLANDRE,
3. 4.
5. 6. 7. 8. 9. 10. 11. 12.
B. A.,
__ Electron-Microscopic Structure of Protozoa. Per&non Press, Oxford, PITELKA, D. R. and SCHOOLEY, C. N., Uniu. Calif. Publ. ZoBf. 61, 79 (1955). PRINGSHEIM, E. G. and HOVASSE, R., Arch. Zool. Expff. Gen. 86, 499 (1950). VLK, W., Arch. Profisfenk. 90, 448 (1938).
Experimenfal
1963.
Cell Research
37