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Ultrastructure of Amastigomonas bermudensis ATCC 50234 sp. nov.
Europ.J.Protistol. 27, 386-396 (1991) November 29, 1991
European Journal of
PROTISTOLOGY
Ultrastructure of Amastigomonas bermudensis ATCC 50234 sp. nov. A New Heterotrophic Marine Flagellate Francis I. Molina and Thomas A. Nerad American Type Culture Collection, Rockville, USA
SUMMARY The ultrastructure of a new marine heterotrophic flagellate is described. The cell is dorso-ventrally flattened and displays a steady gliding forward movement. A longitudinal groove whose lips are appressed to the substrate runs along the ventral aspect of the cell. The two heterodynamic flagella originate from the proximal end of a cytoplasmic sheath that enfolds most of the length of the anterior flagellum. The lips of this sheath are continuous with the margins of the ventral groove. The posterior trailing flagellum is held closely appressed to the cell body on one side of the ventral groove. Three bands of microtubules extend posteriorly into the cell body and are associated with the kinetosomes. The part of the surface membrane extending over the cell's dorsal and lateral aspect is five-layered, giving the semblance of two fused cell membranes. There is a fibrillar layer underneath these membranes. There is also a fibrous network whose arrays are oriented in different directions within the cytoplasm. Mitochondria have tubular cristae. Based on a comparison with previously described species of Amastigomonas, we establish the species, A. bermudensis n.sp. We further conclude that Thecamonas is a junior synonym of Amastigomonas and move the three nominal species to the latter genus.
Introduction
The genus Amastigomonas was established by De Saedeleer in 1931 [11] for a single species which was characterized by the lack of flagella, a gliding movement punctuated by frequent metaboly, and the presence of a "proboscis". In 1971, Zhukov [15] redescribed the genus to include forms with two heterodynamic flagella, one of which was borne on a "proboscis". He established the second species, A. caudata, in 1975 [16]. Later, Hamar [3] described the third species, A. borokiensis. Ultrastructural studies of Amastigomonas have heretofore only been done by Mylnikov [10]. His studies of A. caudata revealed that the whole cell is invested with a five-layered membrane except on the ventral part where feeding takes place. Two heterodynamic flagella are present, both tapering into an acroneme with the anterior one ensheathed by a cytoplasmic fold. The fine structure of A. caudata is reminiscent of Apusomonas proboscidea [16] but Amastigomonas is unique in that it lacks a 0932-4739/91/0027-0386$3.50/0
mastigophore (sensu Vickerman et al. [14]) and has a ventral groove that traverses the entire length of the cell. The uniqueness of these flagellates led Karpoff and Mylnikov [5] to establish the order Apusomonadida within the class Zoomastigophorea to accommodate the genera Apusomonas and Amastigomonas. We report on the ultrastructure of a heterotrophic marine flagellate that is sufficiently distinct from the six most closely related species to warrant assigning it to Amastigomonas bermudensis sp. nov.
Material and Methods Source and isolation ofthe strain. The strain was isolated from a sample collected on 07 October 1984 from Cliff Pool, Bermuda (coordinates: 32°10'N, 64°30'W). It was propagated in ATCC Medium 1525 bacterized with Klebsiella pneumoniae ATCC 27889 and maintained by subculturing into fresh medium every 14-21 d in T-25 tissue culture flasks. © 1991 by Gustav Fischet Vetlag, Stuttgatt
Ultrastructure of Amastigomonas bermudensis . 387 Establishment of clonal cultures. Clonal cultures were established as follows. A small drop of a dilute mixed culture was placed at the edge of a 20 X 100 mm Petri dish containing non-nutrient seawater agar. Bacterized culture medium was slowly added at the opposite end and allowed to slowly engulf the drop. After 30 min, single cells which had dispersed through the culture medium were aspirated with a micropipette and placed in individual 35 mm petri dishes. One of the clonal cultures thus established was chosen and used for these studies. This culture was deposited with the American Type Culture Collection and assigned the accession number ATCC 50234. Light Microscopy. The flagellates attached firmly to the sides of the culture vessel and were difficult to dislodge. It was necessary to agitate the flask vigorously and promptly transfer a drop to a cover slip that had been coated with poly-D-lysine (MW = 150-300 Kd, Sigma Chemical Company Cat. No. P 1149). After five minutes, the cover slip was inverted onto a glass slide and cells were examined with a Zeiss Universal Microscope fitted with phase contrast attachments. Photographs were taken under oil immersion using Kodak Professional T-Max 100 film rated and push-processed at ASA 200. Electron Microscopy. Cells were washed twice with sea water and fixed with an ice-cold mixture of 2% glutaraldehyde and 1% osmium tetroxide in 0.1 M phosphate buffer (pH 7.4) with 10 mM MgCIz and 3.7% (w/v) sodium chloride. The fixative was allowed to warm up to room temperature for 30 min after addition to the cell pack. After a brief wash, cells were post-fixed with 1% osmium tetroxide, washed three times in 5 ml volumes of the vehicle, dehydrated in a graded acerone series, and embedded in PolyBed 812. Silver to pale-gold sections were cut with a DDK diamond knife on a Reichert OMU 2 ultramicrotome and collected on 300-mesh hexagonal copper grids. Sections were stained in methanolic uranyl acetate followed by lead citrate and viewed with a Zeiss EM 10 transmission electron microscope. For Scanning Electron Microscopy (SEM), cells were resuspended in Parducz's fixative [12] consisting of 6 parts 2% osmium tetroxide and 1 part saturated aqueous mercuric chloride. Specimens were fixed for 10 min, washed 12 times with filtersterilized distilled water, and dehydrated through a graded series of ethyl alcohol. After critical point drying, specimens were coated with gold/palladium in a vacuum evaporator (Denton Model DV 503) and viewed with an Amray AMR-lOOO A scanning electron microscope.
Results
Light Microscopy In wet mounts the flagellate was observed to glide slowly along the substrate, its movement often punctuated by brief periods of metaboly. The cell is ovoid and dorsoventrally flattened, with the dorsal part being convex and the ventral part traversed by a groove (Fig. 1). This feature allows one to distinguish between the left and right side of the cell. It is the ventral aspect which is used for firm attachment to the walls of the culture vessel. Once attached to a substratum, the cells are very difficult to dislodge and require vigorous agitation for detachment. Cyst formation was not observed. The flagellate bears two heterodynamic flagella: an actively beating anterior flagellum and a trailing flagellum which resides in one side of the ventral groove (Fig. 3). The latter is held closely appressed to the cell body and is difficult to visualize with the aid of either interference or phase contrast microscopy. It could be seen more clearly during contractions of the body. The anterior flagellum appears thicker than the posterior one because it is ensheathed by a sleeve that originates from the cell body (Figs. 1,2). Its movement is best described as lateral amero-sinistral [14]. It beats back and forth, tracing an acute arc, and the preferred side of beating is always to the left of the cell. There is a collar at the junction of the sleeve and the cell body (Fig. 2). The cell body measures 4.0 fim wide by 9.5 fim long. The sleeve that encloses the base of the anterior flagellum extends over a distance of 4.0 fim, leaving only a short segment (2.0 f.till) of the flagellum's tip uncovered (Figs. 1,2).
Electron Microscopy Scanning electron microscopy confirms the observations made with the phase contrast microscope (Fig. 4). Only a short segment of the anterior flagellum is seen to project
Figs. 1-3. Phase contrast microscopy, X 2500. -Figs. 1, 2. Ventral view of living cells showing the left (LM) and right (RM) marginal folds of the ventral groove. The folds extend anteriorly into a sleeve (arrowhead) that encloses the flagellum. The part of the anterior flagellum that projects beyond the sleeve is visible in both micrographs. The collar (Co) at the base of the sleeve can be seen more clearly in Fig. 2. - Fig. 3. Slightly twisted cell showing the recurrent flagellum (RF) in the ventral groove. - Figs. 4,5. Scanning electron microscopy. - Fig. 4. Ventro-lateral view of whole cell showing the left (LM) and right (RM) marginal folds enclosing the recurrent flagellum (arrowhead), X 13 000. - Fig. 5. Detailed structure of sleeve enclosing the anterior flagellum. Paired knobs (arrowheads) occur at regular intervals along its length, x 24 000. - Figs. 6,7. Transmission electron microscopy, thin sectioning. - Fig. 6. Whole cell under low magnification showing the difference in the thickness of dorsal (DM) and ventral (VM) membranes, X 11000. Fig. 7. Dorsal membrane under high magnification showing the five-layered structure. The middle electron-dense layer is thicker than the exoplasmic (E) and protoplasmic (P) electron-dense layers, X 190 000.
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Figs. 8-12. Transmission electron microscopy, thin sectioning. - Fig. 8. Ingestion of bacteria (B) through the cell's ventral surface, ~ x 24000.-Fig. 9. Structures associated with the plasmalemma: Pe = pellicle, FN = fibrous network, X 45 OOO.-Fig. 10. Wholecell showing the peripheral and central location of the fibrous network. Both longitudinal (arrowheads) and transverse (arrow) sections through the network are present, X 18 000. - Fig. 11. Whole cell with the left marginal fold (LM) enclosing the recurrent flagellum (RF). A microbody-like organelle ('f) is interspersed between two mitochondria, X 23 000. - Fig. 12. Detailed structure of the recurrent flagellum (RF) enclosed by the left marginal fold showing the typical 9 + 2 arrangement of microtubules and no paraxial rod, X 56000.
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390 . F. I. Molina and T. A. Nerad
from the sleeve and its tip does not taper into an acroneme (Fig. 5). A unique structure not detectable using light microscopy is also present: paired knob-like structures which occur at regular intervals on each edge of the sleeve. Under low magnification, the cell is seen to be invested by a very prominent thick surface membrane which runs dorsolaterally and is interrupted at the ventral aspect (Fig. 6). Closer examination of this thick membrane reveals that it is composed of five alternating electrondense and electron-transparent layers (Fig. 7). The middle electron-dense layer is thicker than the exoplasmic (E) and the protoplasmic (P) layers, giving the semblance of two fused unit membranes. The five-layered membrane must terminate at the ventral folds, for the ventral side shows only a unit membrane (Fig. 6). It is in this part of the cell where ingestion of bacteria takes place (Fig. 8). No ornamentations were found on the cell surface. The pellicle (sensu Corliss and Lorn [1]) includes a fibrous layer underlying the cell membrane (Fig. 9). Below this pellicle, there is a fibrillar network whose arrays are oriented in different directions. Individual fibrils comprising the network are spaced about 15 nm apart. That the fibrillar network is distinct from the pellicle is shown by sections where the fibrils are not peripheral and apparently not associated with the pellicle (Fig. 10). The cytoplasm has a well-developed endoplasmic reticulum. In median cross section, the cell exhibits two ventral folds with the left one enclosing the trailing flagellum (Figs. 11 and 12). These folds extend anteriorly to form the sleeve that ensheathes the anterior flagellum (Figs. 13,14). The two flagella originate at the base of the sleeve near its junction with the ventral folds (Fig. 15). Both flagella show the typical 9 + 2 arrangement of axonemal microtubules and have no paraxial rods nor mastigonemes (Fig. 16). There is a single Golgi body near the basal bodies (Fig. 15). In addition, there is a row of reinforcing microtubules on each side of the ventral groove (Fig. 17). The left band of microtubules (LB) consists of 4 microtubules while the right band of microtubules (RB) is composed of 10-12 individual microtubules. A third band of 12-16 microtubules, resembling the multi-layered structure (MLS) found in chlorophytes [9], is associated with the posterior kinetosome. As can be deduced from longitudinal sections (Fig. 18), it apparently extends deep into the cytoplasm. An electron-dense rod (EDR) lies in close proximity to the "MLS" (Figs. 16, 17).
The nucleus is located approximately in the middle of the cell, going from an anterior-posterior direction. It is pushed to one side and has a distinct nucleolus (Fig. 19). Its chromatin is clearly differentiated into eu- and heterochromatin, the latter being commonly associated with the nuclear membrane. The mitochondria have tubular cristae (Fig. 20). Branching of mitochondria was not observed in any of the sections examined. In some sections, microbody-like organelles are interspersed between mitochondria (Fig. 11). Ingestion of substrates is localized and takes place on the ventral aspect of the cell where the surface membrane consists of only a single (unit) layer (Fig. 8). It is here that food vacuoles are formed. Endocytosis appears to occur at several points on the ventral membrane and is not restricted to a single area. Endosomes are formed from invaginations of the plasma membrane, causing the substrate to be internalized. In some sections small vesicles, probably lysosomal in nature, appear to fuse with the endosome (Fig. 21). Figure 22 shows several bacteria that may have been ingested in this fashion. Discussion Definition ofterms. A comparison of zooflagellates that have slender anterior protuberances necessitates standardization of terminology, as these are diagnostic features and different terms have been used for them. The multiplicity of terms is mainly due to the limited information provided by light microscopical techniques that were employed by previous workers. The term "proboscis" has been loosely applied to such a projection in Amastigomonas [3,4]. The same structure was referred to as the "rostrum" by Larsen and Patterson [8]. In our opinion, neither of these terms is appropriate for the structure that encloses the anterior flagellum in Apusomonas and Amastigomonas. We agree with the definition of "proboscis" provided by Corliss and Lorn [1] as "a trunk-like extension of the anterior end of the body of certain ciliates with the organism's oral area at its base (not at its distal extremity)". Therefore, this term should not be used for the anterior projection in these flagellates. With regard to "rostrum", we feel that it should be applied sensu stricto to those structures at the apical end of the cell which are beak-shaped and as such cannot be applied to the structure under consideration.
Figs. 13-17. Transmission electron microscopy, thin sections. - Figs. 13, 14. Transverse (Fig. 13) and longitudinal (Fig. 14) sections ~ through the anterior flagellum showing the axoneme (a), flagellar membrane (arrowhead) and sleeve (51). As in the recurrent flagellum, paraxial rods are absent. Fig. 13, X 53000; Fig. 14, X 35000. - Fig. 15. Origin of anterior (AF) and recurrent flagella (RF). The shaft of the recurrent flagellum is visible while only the kinetosome of the anterior flagellum can be seen. The "multi-layered structure" (") and a Golgi body (G) are also present, X 51000. - Fig. 16. Cross-section through both flagella. The anterior flagellum, easily distinguishable by its enclosing sleeve, has apparently bent over backwards. Both the "multi-layered structure" ("MLS") and its associated electron-dense rod (EDR) are present, X 36000. - Fig. 17. Reinforcing structures of the flagellar system: LB = left band of microtubules, RB = right band of microtubules, * = multilayered structure, and EDR = electron-dense rod, x 46000.
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Ultrastructure of Amastigomonas bermudensis . 393
From the accounts of Vickerman and co-workers [14], it is evident that the ventral folds in Apusomonas proboscidea extend into a mastigophore whose distal end is demarcated by the point of flagellar origin. The marginal folds continue anteriorly from the distal tip of the mastigophore to enclose most of the anterior flagellum. Investigators [5, 14] who have studied A. proboscidea refer to this enclosure as the "sleeve" which we adopt in this paper for a homologous structure in Amastigomonas. The presence or absence of the mastigophore is important in separating members of Apusomonas and Amastigomonas. Light microscopy. According to Larsen and Patterson [8], Amastigomonas might have been confused with members of the genus Rhynchomonas by early workers. Likewise, Hollande [4] stated that the two genera are morphologically analogous except for the lack of a posterior flagellum in the former. We now know that this is incorrect, for a trailing flagellum is present in Amastigomonas but in some species may not be easily seen. Amastigomonas can be distinguished from Rhynchomonas by the presence of a ventral groove in which the trailing flagellum is firmly held. Such a groove was not mentioned in Klebs' [7] original description of the type species, R. nasuta (earlier described as Heteromita nasuta by Stokes [13]). Therefore, it is unlikely that the original description of the genus Rhynchomonas could have been based on a strain of Amastigomonas. Until more extensive studies are done on the genus Amastigomonas, light microscopical characters will have to remain as the primary criteria for delineation of species. These include cell size, flagellar length, length of the flagellar sleeve, and presence/absence of threads emanating from the plasmalemma. Based on the information given in Table 1, we present a dichotomous key (see below) to the seven species that we recognize within genus. Electron microscopy. The fine structure of the organism which we have studied clearly places it in the genus Amastigomonas. There has been only a single detailed account of the ultrastructure of a species within this genus. This species, A. caudata, shares the following characteristics with the new organism: 1) the presence of an ensheathed anterior flagellum which the organism uses for locomotion, 2) a dorso-ventrally flattened cell body with a ventral groove, 3) a five-layered cell membrane on the cell's dorsal and lateral aspects, and 4) mitochondria with tubular cristae. The organism that we describe here has several ultrastructural features that distinguish it from A. caudata. First, the system of microfibrils underlying the cell membrane and distributed as bundles in the cytoplasm has not
been observed in A. caudata. Second, the number of microtubules present in each of the microtubular bands, LB, RB, and "MLS", differs in the two species (Table 1). In particular, the right band and the "MLS" has more microtubules (RB: 10-12 microtubules in our organism and 5 in A. caudata; "MLS": 12-16 in our organism and 8-10 in A. caudata). Third, the anterior flagellum is acronematic in A. caudata and non-acronematic in the other. Finally, the thread-like appendages projecting from the cell membrane are only found in A. caudata [10]. In combination with light microscopical characters (Table 1) such as cell size, formation of "plasmodia", length of flagella, and extent of ensheathment of the anterior flagellum (whether it extends beyond the sleeve - i.e., is emergent), these ultrastructural features clearly separate the new organism we describe from all other species of Amastigomonas. The function of the bundles of fibers in the cytoplasm is unclear. These have not been observed in any species of Amastigomonas nor in Apusomonas proboscidea although in the latter a similar structure, the webnet, is present [6]. It consists of threads emerging from microtubules forming the "MLS". Since the webnet is considered to be a part of the "MLS" [6] and is located close to the nucleus, we do not believe it to be equivalent to the fibrous network. However, differences in fixation methods may preclude direct comparison. The anterior locomotory flagellum of A. bermudensis is ensheathed by an extension of the lips of the ventral groove and beats in a lateral antero-sinistral fashion. Similar observations have been made in A. proboscidea by Vickerman and co-workers [14]. The flagellar sleeve may act to increase the power stroke of the flagellum in much the same manner as mastigonemes. The paired knob-like structures, visualized with scanning electron microscopy, may act as sutures on each edge of this sleeve and may serve to prevent displacement of the anterior flagellum during flagellar beating. However, since these structures were not visualized with either light- or transmission electron microscopy, they may be artifactual. Their preservation may also be dependent on the type of fixation used. Hence, further studies are needed to establish whether or not these knobs are regular features of the cell. The structure of the new organism, deduced from light and electron microscopy, is depicted in Fig. 23. The differences between it and other described species of Amastigomonas are summarized in Table 1. These features are used as bases for the erection of a new species. Based on evidence from light and electron microscopy (TEM and SEM), Amastigomonas bermudensis sp. nov. is created. However, because the origins of the microtubular
• Figs. 18-22. Transmission electron microscopy, thin sections. - Fig. 18. Longitudinal section through the "multi-layered structure" ("MLS"), X 36000. - Fig. 19. Nucleus with prominent nucleolus (Nu) and heterochromatin, x 26000. - Fig. 20. Mitochondrion with tubular cristae. Thefive-layered structure ofthe surface membrane is also evident, X 79000. - Fig. 21. Apparentfusion of vesicles (v) with a food vacuole (FV). These vesicles may deliver lysosomal enzymes, x 102000. - Fig. 22. Food vacuoles containing ingested bacteria (arrowheads), X 38000.
394 . F. 1. Molina and T. A. Nerad
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23 Fig. 23. Morphology of A. bermudensis, deduced from light and electron microscopy. Cross-sections are shown to the right of the appropriate level. The top cross-section has been enlarged five times relative to the rest of the cell to show fine detail.
Ultrastructure of Amastigomonas bermudensis . 395 Table 1. Summary of differences between A. bermudensis sp. nov. and other species of Amastigomonas Reference
A. bermuden- A. borokiensis A. caudata [10] sis [3] This paper
1. Cell dimensions a. Length b. Width
8.0-11.5 Jlm 5.0-9.0 Jlm 3.0-5.5 Jlm 3.0-4.0/lm
4.0-5.0 Jlm 2.0 Jlm
A. debruynei [11]
A. mutabilis [2]
A. filosa [8]
A. trahens [8]
5.0/lm 2.8 Jlm 2.0 Ilm<'
9.0-14.0 Jlm 4.0 Jlm
5.5-7.0/lm NR
4.5-7.0 Jlm 3.0-5.0 Jlm
2.5 Ilm"
2.0 Jlm
NR
5.0-7.0 Jlm
Variable
+
NR
2. Length of flagellar 4.0 Jlm sleeve
2.0/lm*
3. Length of anterior 2.0 Jlm flagellum projecting from the sleeve
Non-emergent Non-emetgent Non-emergent 3.0-4.0 Jlm
4. Flagellar acroneme
-
NR
+
NR
+
5. Sub-plasmalemmal fibrous network
+
NR
-
NR
NR
6. Thread-like append ages on cell surface
-
NR
+
NR
-
7. Electron-dense rod + adjacent to MLS
NR
+
NR
NR
NR NR NR
3-5 5 8-10
NR NR NR
NR NR NR
NR NR NR
NR NR NR
Freshwater
Marine
Freshwater
Marine
Marine
Marine
8. No. of microtubuies in microtubular bands: Left 4 10-12 Right "MLS" 12-16 10. Habitat NR = Not reported.
Marine
0.8-1.0 Jlm
+
* = Estimated from drawings in the original manuscript.
ribbons have not been traced back to the kinetosomes, a precise phylogenetic analysis is not feasible at this time. Until further studies are done, we are adopting the taxonomic treatment of Karpoff and Mylnikov [5].
Taxonomic Diagnosis Amastigomonas bermudensis Molina et Nerad sp. nov. Biflagellate heterotrophic protists with a ventral groove whose marginal folds extend anteriorly forming a sleeve that encloses the anterior locomotory flagellum. Flagella smooth and non-acronematous, originating at the base of the sleeve. Beating of anterior flagellum lateral anterosinistral, tracing an acute arc. Cells gliding on the substrate and feeding on bacteria. Recurrent flagellum located in the left side of the ventral groove and held close to the cell surface. Cell dorso-ventrally flattened. Dorsal surface convex, covered by a five-layered membrane. Ventral groove used for ingestion of bacteria and marginal folds firmly appressed to the substratum. Mitochondria with tubular cristae. Golgi apparatus present. Three bands of microtubules associated with the kinetosomes. One of these, the "MLS", shows an adjacent electron-dense,
rod-like body. Placed in the order Apusomonadida, class Zoomastigophorea. Single cells 4.2 ± 0.7 j.,lm wide (range: 3.0-5.5 j.,lm) X 9.5 ± 1.0 j.,lm long (range: 8.0-11.5 j.,lm). Flagellar sheath 4.1 ± 0.4 j.,lm (range: 3.5-4.5 !lm). Type material: Collected in detritus from Cliff Pool, Bermuda (coordinates: 32°10'N, 64°30'W), October 1984. Deposited with the American Type Culture Collection under accession number ATCC 50234.
New Combinations Larsen and Patterson [8] created the genus Thecamonas and provided the following description: "Gliding flagellates, with one laterally or anteriorly directed flagellum, one flagellum trailing under left margin of the cell. Body plastic. Dorsal surface and base of anterior flagellum with a pliable organic sheath (theca)". These characters are exhibited by the genus Amastigomonas, described earlier by De Saedeleer [11] and more recently by Zhukov [15, 16]. The ultrastructure of the type species of Thecamonas, T. trahens (Patterson, personal communication) and T. mutabilis [8], parallels that of Amastigomonas as reported by Mylnikov [10]. The two genera share the following characteristics: 1) a partly ensheathed anterior flagellum, 2) a trailing flagellum that lies on the left side of
396 . F. I. Molina and T. A. Nerad
the ventral groove, 3) dorsal and lateral surfaces invested by a multi-layered membrane that becomes a unit membrane in the ventral groove, 4) mitochondria with tubular cristae, and 5) three distinct microtubular ribbons. Because the two genera are indistinguishable and Amastigomonas has priority over Thecamonas, the latter must be treated as a junior synonym. Therefore, the following new combinations are necessary:
Amastigomonas mutabilis (Griessman, 1913) Molina et Nerad comb. nov. Basionym: Rhynchomonas mutabilis Griessman, 1913. Synonym: Thecamonas mutabilis Larsen and Patterson, 1990. With characters of the genus. Amastigomonas trahens (Larsen and Patterson 1990) Molina et Nerad comb. nov. Synonym: Thecamonas trahens Larsen and Patterson, 1990. With characters of the genus. Amastigomonas filosa (Larsen and Patterson 1990) Molina et Nerad comb. nov. Synonym: Thecamonas filosa Larsen and Patterson, 1990. With characters of the genus. Key to Species of Amastigomonas 1a
Thread-like appendages present on cell surface 1b Thread-like appendages absent 2a Anterior flagellum emergent from sleeve, cells 5.5-7.0 !tm 2b Anterior flagellum non-emergent, cells 4.0-5.0 !tm 3a Anterior flagellum emergent from sleeve 3a Anterior flagellum non-emergent 4a Cells under 8.0 !tm in length 4b Cells over 8.0 !tm in length 5a Cells not exceeding 5.0 !tm in length 5b Cells between 5.0 and 9.0 !tm in length 6a Flagellar sleeve 4.0 !tm in length, anterior flagellum projecting 2.0 !tm from the sleeve 6b Flagellar sleeve 2.5 !tm in length, anterior flagellum projecting 3.0-4.0 !tm from the sleeve
2
3 A. filosa A. caudata
4 5 A. 6 A. A. A.
trahens debruynei borokiensis bermudensis
A. mutabilis
Acknowledgements This study was supported in part by NSF grant BSR 8514344. We wish to thank Tim Maugel, Laboratory for Biological Ultrastructure, University of Maryland (College Park), for use of
electron microscopy facilities and Lisa Allen for the illustration. We thank Dr. Eugene B. Small of the University of Maryland for collecting the sample and depositing the strain at the ATCC.
References 1 Corliss J. O. and Lorn J. (1984): An annotated glossary of protozoological terms. In: Lee J. J., Hutner S. H. and Bovee E. C. (ed.): An illustrated guide to the protozoa, pp. 576-602. Society of Protozoologists, Lawrence, Kansas. 2 Griesmann K. (1913): Dber marine Flagellaten. Arch. Protistenkd., 32, 1-78. 3 Hamar J. (1979): Some new zooflagellates from Hungary. Tiscia, 14, 147-162. 4 Hollande A. (1952): Ordre des bodonides. In: Grasse P. P. (ed.): Traite de Zoologie. Anatomie-systematique biologie. Tome I, Fascicule I, Protozaires (generalites, flagelles), pp. 669-693. Masson, Paris. 5 Karpoff S. A. and Mylnikov A. P. (1989): Biology and ultrastructure of colorless flagellates Apusomonadida ord. n. Zool. Zh., 68, 8-17. 6 Karpoff S. A. and Zhukov B. F. (1986): Ultrastructure and taxonomic position of Apusomonas proboscidea. Arch. Protistenkd., 131, 13-26. 7 Klebs G. (1892): Dber die Organisation einiger Flagellatengruppen und ihre Beziehungen zu Algen und Infusorien. Untersuchungen Botanisches Institut Tiibingen, 1, 233-362. 8 Larsen J. and Patterson D. J. (1990): Some flagellates (Protista) from tropical marine sediments. J. Nat. Hist., 24, 801-937. 9 Moestrup 0. (1982): Flagellar structure in algae: a review, with new observations particularly on the Chrysophyceae, Phaeophyceae (Fucophyceae), Euglenophyceae, and Reckertia. Phycologia, 21, 427-528. 10 Mylnikov A. P. (1989): The ultrastructure of the flagellate Amastigomonas caudata. Tsiitologia, 31, 489-491 (In Russian). 11 Saedeleer H. De. (1931): Nieuwe of weinig bekende Flagellaten. Natuurweten-schappelijk, Tidjschrift, 13, 3-5. 12 Small E. B. and Marszalek D. S. (1970): Scanning electron microscopy of fixed, frozen, and dried protozoa. Science, 163, 1064-1065. 13 Stokes A. C. (1888): Notices of new infusoria flagellata from American fresh waters. J. R. Microsc. Soc. 3rd Series, 8, 698-704. 14 Vickerman K., Darbyshire J. F. and Ogden C. G. (1974): Apusomonas proboscidea Alexeieff 1924 an unusual phagotrophic flagellate from soil. Arch. Protistenkd., 116, 254-269. 15 Zhukov B. F. (1971): Keys to the colorless free-living Flagellata of the suborder Bodonida. Biology and productivity of freshwater organisms. Nauka, 1971, 241-284. 16 Zhukov B. F. (1975): Amastigomonas caudata sp. n. (suborder Bodonina Holl., order Kinetoplastida Honigberg, class Zoomastigophorea Calkins, Protozoa). Academy of Sciences of the USSR. Biology of Inland Waters Information Bull., 19, 25-26.
Key words: Ultrastructure - Taxonomy - Flagellate - Amastigomonas - Apusomonas Francis I. Molina, American Type Culture Collection, 12301 Parklawn Drive, Rockville 20852, MD, USA
European Journal of
PROTISTOLOGY
Ultrastructure of Amastigomonas bermudensis ATCC 50234 sp. nov. A New Heterotrophic Marine Flagellate Francis I. Molina and Thomas A. Nerad American Type Culture Collection, Rockville, USA
SUMMARY The ultrastructure of a new marine heterotrophic flagellate is described. The cell is dorso-ventrally flattened and displays a steady gliding forward movement. A longitudinal groove whose lips are appressed to the substrate runs along the ventral aspect of the cell. The two heterodynamic flagella originate from the proximal end of a cytoplasmic sheath that enfolds most of the length of the anterior flagellum. The lips of this sheath are continuous with the margins of the ventral groove. The posterior trailing flagellum is held closely appressed to the cell body on one side of the ventral groove. Three bands of microtubules extend posteriorly into the cell body and are associated with the kinetosomes. The part of the surface membrane extending over the cell's dorsal and lateral aspect is five-layered, giving the semblance of two fused cell membranes. There is a fibrillar layer underneath these membranes. There is also a fibrous network whose arrays are oriented in different directions within the cytoplasm. Mitochondria have tubular cristae. Based on a comparison with previously described species of Amastigomonas, we establish the species, A. bermudensis n.sp. We further conclude that Thecamonas is a junior synonym of Amastigomonas and move the three nominal species to the latter genus.
Introduction
The genus Amastigomonas was established by De Saedeleer in 1931 [11] for a single species which was characterized by the lack of flagella, a gliding movement punctuated by frequent metaboly, and the presence of a "proboscis". In 1971, Zhukov [15] redescribed the genus to include forms with two heterodynamic flagella, one of which was borne on a "proboscis". He established the second species, A. caudata, in 1975 [16]. Later, Hamar [3] described the third species, A. borokiensis. Ultrastructural studies of Amastigomonas have heretofore only been done by Mylnikov [10]. His studies of A. caudata revealed that the whole cell is invested with a five-layered membrane except on the ventral part where feeding takes place. Two heterodynamic flagella are present, both tapering into an acroneme with the anterior one ensheathed by a cytoplasmic fold. The fine structure of A. caudata is reminiscent of Apusomonas proboscidea [16] but Amastigomonas is unique in that it lacks a 0932-4739/91/0027-0386$3.50/0
mastigophore (sensu Vickerman et al. [14]) and has a ventral groove that traverses the entire length of the cell. The uniqueness of these flagellates led Karpoff and Mylnikov [5] to establish the order Apusomonadida within the class Zoomastigophorea to accommodate the genera Apusomonas and Amastigomonas. We report on the ultrastructure of a heterotrophic marine flagellate that is sufficiently distinct from the six most closely related species to warrant assigning it to Amastigomonas bermudensis sp. nov.
Material and Methods Source and isolation ofthe strain. The strain was isolated from a sample collected on 07 October 1984 from Cliff Pool, Bermuda (coordinates: 32°10'N, 64°30'W). It was propagated in ATCC Medium 1525 bacterized with Klebsiella pneumoniae ATCC 27889 and maintained by subculturing into fresh medium every 14-21 d in T-25 tissue culture flasks. © 1991 by Gustav Fischet Vetlag, Stuttgatt
Ultrastructure of Amastigomonas bermudensis . 387 Establishment of clonal cultures. Clonal cultures were established as follows. A small drop of a dilute mixed culture was placed at the edge of a 20 X 100 mm Petri dish containing non-nutrient seawater agar. Bacterized culture medium was slowly added at the opposite end and allowed to slowly engulf the drop. After 30 min, single cells which had dispersed through the culture medium were aspirated with a micropipette and placed in individual 35 mm petri dishes. One of the clonal cultures thus established was chosen and used for these studies. This culture was deposited with the American Type Culture Collection and assigned the accession number ATCC 50234. Light Microscopy. The flagellates attached firmly to the sides of the culture vessel and were difficult to dislodge. It was necessary to agitate the flask vigorously and promptly transfer a drop to a cover slip that had been coated with poly-D-lysine (MW = 150-300 Kd, Sigma Chemical Company Cat. No. P 1149). After five minutes, the cover slip was inverted onto a glass slide and cells were examined with a Zeiss Universal Microscope fitted with phase contrast attachments. Photographs were taken under oil immersion using Kodak Professional T-Max 100 film rated and push-processed at ASA 200. Electron Microscopy. Cells were washed twice with sea water and fixed with an ice-cold mixture of 2% glutaraldehyde and 1% osmium tetroxide in 0.1 M phosphate buffer (pH 7.4) with 10 mM MgCIz and 3.7% (w/v) sodium chloride. The fixative was allowed to warm up to room temperature for 30 min after addition to the cell pack. After a brief wash, cells were post-fixed with 1% osmium tetroxide, washed three times in 5 ml volumes of the vehicle, dehydrated in a graded acerone series, and embedded in PolyBed 812. Silver to pale-gold sections were cut with a DDK diamond knife on a Reichert OMU 2 ultramicrotome and collected on 300-mesh hexagonal copper grids. Sections were stained in methanolic uranyl acetate followed by lead citrate and viewed with a Zeiss EM 10 transmission electron microscope. For Scanning Electron Microscopy (SEM), cells were resuspended in Parducz's fixative [12] consisting of 6 parts 2% osmium tetroxide and 1 part saturated aqueous mercuric chloride. Specimens were fixed for 10 min, washed 12 times with filtersterilized distilled water, and dehydrated through a graded series of ethyl alcohol. After critical point drying, specimens were coated with gold/palladium in a vacuum evaporator (Denton Model DV 503) and viewed with an Amray AMR-lOOO A scanning electron microscope.
Results
Light Microscopy In wet mounts the flagellate was observed to glide slowly along the substrate, its movement often punctuated by brief periods of metaboly. The cell is ovoid and dorsoventrally flattened, with the dorsal part being convex and the ventral part traversed by a groove (Fig. 1). This feature allows one to distinguish between the left and right side of the cell. It is the ventral aspect which is used for firm attachment to the walls of the culture vessel. Once attached to a substratum, the cells are very difficult to dislodge and require vigorous agitation for detachment. Cyst formation was not observed. The flagellate bears two heterodynamic flagella: an actively beating anterior flagellum and a trailing flagellum which resides in one side of the ventral groove (Fig. 3). The latter is held closely appressed to the cell body and is difficult to visualize with the aid of either interference or phase contrast microscopy. It could be seen more clearly during contractions of the body. The anterior flagellum appears thicker than the posterior one because it is ensheathed by a sleeve that originates from the cell body (Figs. 1,2). Its movement is best described as lateral amero-sinistral [14]. It beats back and forth, tracing an acute arc, and the preferred side of beating is always to the left of the cell. There is a collar at the junction of the sleeve and the cell body (Fig. 2). The cell body measures 4.0 fim wide by 9.5 fim long. The sleeve that encloses the base of the anterior flagellum extends over a distance of 4.0 fim, leaving only a short segment (2.0 f.till) of the flagellum's tip uncovered (Figs. 1,2).
Electron Microscopy Scanning electron microscopy confirms the observations made with the phase contrast microscope (Fig. 4). Only a short segment of the anterior flagellum is seen to project
Figs. 1-3. Phase contrast microscopy, X 2500. -Figs. 1, 2. Ventral view of living cells showing the left (LM) and right (RM) marginal folds of the ventral groove. The folds extend anteriorly into a sleeve (arrowhead) that encloses the flagellum. The part of the anterior flagellum that projects beyond the sleeve is visible in both micrographs. The collar (Co) at the base of the sleeve can be seen more clearly in Fig. 2. - Fig. 3. Slightly twisted cell showing the recurrent flagellum (RF) in the ventral groove. - Figs. 4,5. Scanning electron microscopy. - Fig. 4. Ventro-lateral view of whole cell showing the left (LM) and right (RM) marginal folds enclosing the recurrent flagellum (arrowhead), X 13 000. - Fig. 5. Detailed structure of sleeve enclosing the anterior flagellum. Paired knobs (arrowheads) occur at regular intervals along its length, x 24 000. - Figs. 6,7. Transmission electron microscopy, thin sectioning. - Fig. 6. Whole cell under low magnification showing the difference in the thickness of dorsal (DM) and ventral (VM) membranes, X 11000. Fig. 7. Dorsal membrane under high magnification showing the five-layered structure. The middle electron-dense layer is thicker than the exoplasmic (E) and protoplasmic (P) electron-dense layers, X 190 000.
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Figs. 8-12. Transmission electron microscopy, thin sectioning. - Fig. 8. Ingestion of bacteria (B) through the cell's ventral surface, ~ x 24000.-Fig. 9. Structures associated with the plasmalemma: Pe = pellicle, FN = fibrous network, X 45 OOO.-Fig. 10. Wholecell showing the peripheral and central location of the fibrous network. Both longitudinal (arrowheads) and transverse (arrow) sections through the network are present, X 18 000. - Fig. 11. Whole cell with the left marginal fold (LM) enclosing the recurrent flagellum (RF). A microbody-like organelle ('f) is interspersed between two mitochondria, X 23 000. - Fig. 12. Detailed structure of the recurrent flagellum (RF) enclosed by the left marginal fold showing the typical 9 + 2 arrangement of microtubules and no paraxial rod, X 56000.
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390 . F. I. Molina and T. A. Nerad
from the sleeve and its tip does not taper into an acroneme (Fig. 5). A unique structure not detectable using light microscopy is also present: paired knob-like structures which occur at regular intervals on each edge of the sleeve. Under low magnification, the cell is seen to be invested by a very prominent thick surface membrane which runs dorsolaterally and is interrupted at the ventral aspect (Fig. 6). Closer examination of this thick membrane reveals that it is composed of five alternating electrondense and electron-transparent layers (Fig. 7). The middle electron-dense layer is thicker than the exoplasmic (E) and the protoplasmic (P) layers, giving the semblance of two fused unit membranes. The five-layered membrane must terminate at the ventral folds, for the ventral side shows only a unit membrane (Fig. 6). It is in this part of the cell where ingestion of bacteria takes place (Fig. 8). No ornamentations were found on the cell surface. The pellicle (sensu Corliss and Lorn [1]) includes a fibrous layer underlying the cell membrane (Fig. 9). Below this pellicle, there is a fibrillar network whose arrays are oriented in different directions. Individual fibrils comprising the network are spaced about 15 nm apart. That the fibrillar network is distinct from the pellicle is shown by sections where the fibrils are not peripheral and apparently not associated with the pellicle (Fig. 10). The cytoplasm has a well-developed endoplasmic reticulum. In median cross section, the cell exhibits two ventral folds with the left one enclosing the trailing flagellum (Figs. 11 and 12). These folds extend anteriorly to form the sleeve that ensheathes the anterior flagellum (Figs. 13,14). The two flagella originate at the base of the sleeve near its junction with the ventral folds (Fig. 15). Both flagella show the typical 9 + 2 arrangement of axonemal microtubules and have no paraxial rods nor mastigonemes (Fig. 16). There is a single Golgi body near the basal bodies (Fig. 15). In addition, there is a row of reinforcing microtubules on each side of the ventral groove (Fig. 17). The left band of microtubules (LB) consists of 4 microtubules while the right band of microtubules (RB) is composed of 10-12 individual microtubules. A third band of 12-16 microtubules, resembling the multi-layered structure (MLS) found in chlorophytes [9], is associated with the posterior kinetosome. As can be deduced from longitudinal sections (Fig. 18), it apparently extends deep into the cytoplasm. An electron-dense rod (EDR) lies in close proximity to the "MLS" (Figs. 16, 17).
The nucleus is located approximately in the middle of the cell, going from an anterior-posterior direction. It is pushed to one side and has a distinct nucleolus (Fig. 19). Its chromatin is clearly differentiated into eu- and heterochromatin, the latter being commonly associated with the nuclear membrane. The mitochondria have tubular cristae (Fig. 20). Branching of mitochondria was not observed in any of the sections examined. In some sections, microbody-like organelles are interspersed between mitochondria (Fig. 11). Ingestion of substrates is localized and takes place on the ventral aspect of the cell where the surface membrane consists of only a single (unit) layer (Fig. 8). It is here that food vacuoles are formed. Endocytosis appears to occur at several points on the ventral membrane and is not restricted to a single area. Endosomes are formed from invaginations of the plasma membrane, causing the substrate to be internalized. In some sections small vesicles, probably lysosomal in nature, appear to fuse with the endosome (Fig. 21). Figure 22 shows several bacteria that may have been ingested in this fashion. Discussion Definition ofterms. A comparison of zooflagellates that have slender anterior protuberances necessitates standardization of terminology, as these are diagnostic features and different terms have been used for them. The multiplicity of terms is mainly due to the limited information provided by light microscopical techniques that were employed by previous workers. The term "proboscis" has been loosely applied to such a projection in Amastigomonas [3,4]. The same structure was referred to as the "rostrum" by Larsen and Patterson [8]. In our opinion, neither of these terms is appropriate for the structure that encloses the anterior flagellum in Apusomonas and Amastigomonas. We agree with the definition of "proboscis" provided by Corliss and Lorn [1] as "a trunk-like extension of the anterior end of the body of certain ciliates with the organism's oral area at its base (not at its distal extremity)". Therefore, this term should not be used for the anterior projection in these flagellates. With regard to "rostrum", we feel that it should be applied sensu stricto to those structures at the apical end of the cell which are beak-shaped and as such cannot be applied to the structure under consideration.
Figs. 13-17. Transmission electron microscopy, thin sections. - Figs. 13, 14. Transverse (Fig. 13) and longitudinal (Fig. 14) sections ~ through the anterior flagellum showing the axoneme (a), flagellar membrane (arrowhead) and sleeve (51). As in the recurrent flagellum, paraxial rods are absent. Fig. 13, X 53000; Fig. 14, X 35000. - Fig. 15. Origin of anterior (AF) and recurrent flagella (RF). The shaft of the recurrent flagellum is visible while only the kinetosome of the anterior flagellum can be seen. The "multi-layered structure" (") and a Golgi body (G) are also present, X 51000. - Fig. 16. Cross-section through both flagella. The anterior flagellum, easily distinguishable by its enclosing sleeve, has apparently bent over backwards. Both the "multi-layered structure" ("MLS") and its associated electron-dense rod (EDR) are present, X 36000. - Fig. 17. Reinforcing structures of the flagellar system: LB = left band of microtubules, RB = right band of microtubules, * = multilayered structure, and EDR = electron-dense rod, x 46000.
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Ultrastructure of Amastigomonas bermudensis . 393
From the accounts of Vickerman and co-workers [14], it is evident that the ventral folds in Apusomonas proboscidea extend into a mastigophore whose distal end is demarcated by the point of flagellar origin. The marginal folds continue anteriorly from the distal tip of the mastigophore to enclose most of the anterior flagellum. Investigators [5, 14] who have studied A. proboscidea refer to this enclosure as the "sleeve" which we adopt in this paper for a homologous structure in Amastigomonas. The presence or absence of the mastigophore is important in separating members of Apusomonas and Amastigomonas. Light microscopy. According to Larsen and Patterson [8], Amastigomonas might have been confused with members of the genus Rhynchomonas by early workers. Likewise, Hollande [4] stated that the two genera are morphologically analogous except for the lack of a posterior flagellum in the former. We now know that this is incorrect, for a trailing flagellum is present in Amastigomonas but in some species may not be easily seen. Amastigomonas can be distinguished from Rhynchomonas by the presence of a ventral groove in which the trailing flagellum is firmly held. Such a groove was not mentioned in Klebs' [7] original description of the type species, R. nasuta (earlier described as Heteromita nasuta by Stokes [13]). Therefore, it is unlikely that the original description of the genus Rhynchomonas could have been based on a strain of Amastigomonas. Until more extensive studies are done on the genus Amastigomonas, light microscopical characters will have to remain as the primary criteria for delineation of species. These include cell size, flagellar length, length of the flagellar sleeve, and presence/absence of threads emanating from the plasmalemma. Based on the information given in Table 1, we present a dichotomous key (see below) to the seven species that we recognize within genus. Electron microscopy. The fine structure of the organism which we have studied clearly places it in the genus Amastigomonas. There has been only a single detailed account of the ultrastructure of a species within this genus. This species, A. caudata, shares the following characteristics with the new organism: 1) the presence of an ensheathed anterior flagellum which the organism uses for locomotion, 2) a dorso-ventrally flattened cell body with a ventral groove, 3) a five-layered cell membrane on the cell's dorsal and lateral aspects, and 4) mitochondria with tubular cristae. The organism that we describe here has several ultrastructural features that distinguish it from A. caudata. First, the system of microfibrils underlying the cell membrane and distributed as bundles in the cytoplasm has not
been observed in A. caudata. Second, the number of microtubules present in each of the microtubular bands, LB, RB, and "MLS", differs in the two species (Table 1). In particular, the right band and the "MLS" has more microtubules (RB: 10-12 microtubules in our organism and 5 in A. caudata; "MLS": 12-16 in our organism and 8-10 in A. caudata). Third, the anterior flagellum is acronematic in A. caudata and non-acronematic in the other. Finally, the thread-like appendages projecting from the cell membrane are only found in A. caudata [10]. In combination with light microscopical characters (Table 1) such as cell size, formation of "plasmodia", length of flagella, and extent of ensheathment of the anterior flagellum (whether it extends beyond the sleeve - i.e., is emergent), these ultrastructural features clearly separate the new organism we describe from all other species of Amastigomonas. The function of the bundles of fibers in the cytoplasm is unclear. These have not been observed in any species of Amastigomonas nor in Apusomonas proboscidea although in the latter a similar structure, the webnet, is present [6]. It consists of threads emerging from microtubules forming the "MLS". Since the webnet is considered to be a part of the "MLS" [6] and is located close to the nucleus, we do not believe it to be equivalent to the fibrous network. However, differences in fixation methods may preclude direct comparison. The anterior locomotory flagellum of A. bermudensis is ensheathed by an extension of the lips of the ventral groove and beats in a lateral antero-sinistral fashion. Similar observations have been made in A. proboscidea by Vickerman and co-workers [14]. The flagellar sleeve may act to increase the power stroke of the flagellum in much the same manner as mastigonemes. The paired knob-like structures, visualized with scanning electron microscopy, may act as sutures on each edge of this sleeve and may serve to prevent displacement of the anterior flagellum during flagellar beating. However, since these structures were not visualized with either light- or transmission electron microscopy, they may be artifactual. Their preservation may also be dependent on the type of fixation used. Hence, further studies are needed to establish whether or not these knobs are regular features of the cell. The structure of the new organism, deduced from light and electron microscopy, is depicted in Fig. 23. The differences between it and other described species of Amastigomonas are summarized in Table 1. These features are used as bases for the erection of a new species. Based on evidence from light and electron microscopy (TEM and SEM), Amastigomonas bermudensis sp. nov. is created. However, because the origins of the microtubular
• Figs. 18-22. Transmission electron microscopy, thin sections. - Fig. 18. Longitudinal section through the "multi-layered structure" ("MLS"), X 36000. - Fig. 19. Nucleus with prominent nucleolus (Nu) and heterochromatin, x 26000. - Fig. 20. Mitochondrion with tubular cristae. Thefive-layered structure ofthe surface membrane is also evident, X 79000. - Fig. 21. Apparentfusion of vesicles (v) with a food vacuole (FV). These vesicles may deliver lysosomal enzymes, x 102000. - Fig. 22. Food vacuoles containing ingested bacteria (arrowheads), X 38000.
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23 Fig. 23. Morphology of A. bermudensis, deduced from light and electron microscopy. Cross-sections are shown to the right of the appropriate level. The top cross-section has been enlarged five times relative to the rest of the cell to show fine detail.
Ultrastructure of Amastigomonas bermudensis . 395 Table 1. Summary of differences between A. bermudensis sp. nov. and other species of Amastigomonas Reference
A. bermuden- A. borokiensis A. caudata [10] sis [3] This paper
1. Cell dimensions a. Length b. Width
8.0-11.5 Jlm 5.0-9.0 Jlm 3.0-5.5 Jlm 3.0-4.0/lm
4.0-5.0 Jlm 2.0 Jlm
A. debruynei [11]
A. mutabilis [2]
A. filosa [8]
A. trahens [8]
5.0/lm 2.8 Jlm 2.0 Ilm<'
9.0-14.0 Jlm 4.0 Jlm
5.5-7.0/lm NR
4.5-7.0 Jlm 3.0-5.0 Jlm
2.5 Ilm"
2.0 Jlm
NR
5.0-7.0 Jlm
Variable
+
NR
2. Length of flagellar 4.0 Jlm sleeve
2.0/lm*
3. Length of anterior 2.0 Jlm flagellum projecting from the sleeve
Non-emergent Non-emetgent Non-emergent 3.0-4.0 Jlm
4. Flagellar acroneme
-
NR
+
NR
+
5. Sub-plasmalemmal fibrous network
+
NR
-
NR
NR
6. Thread-like append ages on cell surface
-
NR
+
NR
-
7. Electron-dense rod + adjacent to MLS
NR
+
NR
NR
NR NR NR
3-5 5 8-10
NR NR NR
NR NR NR
NR NR NR
NR NR NR
Freshwater
Marine
Freshwater
Marine
Marine
Marine
8. No. of microtubuies in microtubular bands: Left 4 10-12 Right "MLS" 12-16 10. Habitat NR = Not reported.
Marine
0.8-1.0 Jlm
+
* = Estimated from drawings in the original manuscript.
ribbons have not been traced back to the kinetosomes, a precise phylogenetic analysis is not feasible at this time. Until further studies are done, we are adopting the taxonomic treatment of Karpoff and Mylnikov [5].
Taxonomic Diagnosis Amastigomonas bermudensis Molina et Nerad sp. nov. Biflagellate heterotrophic protists with a ventral groove whose marginal folds extend anteriorly forming a sleeve that encloses the anterior locomotory flagellum. Flagella smooth and non-acronematous, originating at the base of the sleeve. Beating of anterior flagellum lateral anterosinistral, tracing an acute arc. Cells gliding on the substrate and feeding on bacteria. Recurrent flagellum located in the left side of the ventral groove and held close to the cell surface. Cell dorso-ventrally flattened. Dorsal surface convex, covered by a five-layered membrane. Ventral groove used for ingestion of bacteria and marginal folds firmly appressed to the substratum. Mitochondria with tubular cristae. Golgi apparatus present. Three bands of microtubules associated with the kinetosomes. One of these, the "MLS", shows an adjacent electron-dense,
rod-like body. Placed in the order Apusomonadida, class Zoomastigophorea. Single cells 4.2 ± 0.7 j.,lm wide (range: 3.0-5.5 j.,lm) X 9.5 ± 1.0 j.,lm long (range: 8.0-11.5 j.,lm). Flagellar sheath 4.1 ± 0.4 j.,lm (range: 3.5-4.5 !lm). Type material: Collected in detritus from Cliff Pool, Bermuda (coordinates: 32°10'N, 64°30'W), October 1984. Deposited with the American Type Culture Collection under accession number ATCC 50234.
New Combinations Larsen and Patterson [8] created the genus Thecamonas and provided the following description: "Gliding flagellates, with one laterally or anteriorly directed flagellum, one flagellum trailing under left margin of the cell. Body plastic. Dorsal surface and base of anterior flagellum with a pliable organic sheath (theca)". These characters are exhibited by the genus Amastigomonas, described earlier by De Saedeleer [11] and more recently by Zhukov [15, 16]. The ultrastructure of the type species of Thecamonas, T. trahens (Patterson, personal communication) and T. mutabilis [8], parallels that of Amastigomonas as reported by Mylnikov [10]. The two genera share the following characteristics: 1) a partly ensheathed anterior flagellum, 2) a trailing flagellum that lies on the left side of
396 . F. I. Molina and T. A. Nerad
the ventral groove, 3) dorsal and lateral surfaces invested by a multi-layered membrane that becomes a unit membrane in the ventral groove, 4) mitochondria with tubular cristae, and 5) three distinct microtubular ribbons. Because the two genera are indistinguishable and Amastigomonas has priority over Thecamonas, the latter must be treated as a junior synonym. Therefore, the following new combinations are necessary:
Amastigomonas mutabilis (Griessman, 1913) Molina et Nerad comb. nov. Basionym: Rhynchomonas mutabilis Griessman, 1913. Synonym: Thecamonas mutabilis Larsen and Patterson, 1990. With characters of the genus. Amastigomonas trahens (Larsen and Patterson 1990) Molina et Nerad comb. nov. Synonym: Thecamonas trahens Larsen and Patterson, 1990. With characters of the genus. Amastigomonas filosa (Larsen and Patterson 1990) Molina et Nerad comb. nov. Synonym: Thecamonas filosa Larsen and Patterson, 1990. With characters of the genus. Key to Species of Amastigomonas 1a
Thread-like appendages present on cell surface 1b Thread-like appendages absent 2a Anterior flagellum emergent from sleeve, cells 5.5-7.0 !tm 2b Anterior flagellum non-emergent, cells 4.0-5.0 !tm 3a Anterior flagellum emergent from sleeve 3a Anterior flagellum non-emergent 4a Cells under 8.0 !tm in length 4b Cells over 8.0 !tm in length 5a Cells not exceeding 5.0 !tm in length 5b Cells between 5.0 and 9.0 !tm in length 6a Flagellar sleeve 4.0 !tm in length, anterior flagellum projecting 2.0 !tm from the sleeve 6b Flagellar sleeve 2.5 !tm in length, anterior flagellum projecting 3.0-4.0 !tm from the sleeve
2
3 A. filosa A. caudata
4 5 A. 6 A. A. A.
trahens debruynei borokiensis bermudensis
A. mutabilis
Acknowledgements This study was supported in part by NSF grant BSR 8514344. We wish to thank Tim Maugel, Laboratory for Biological Ultrastructure, University of Maryland (College Park), for use of
electron microscopy facilities and Lisa Allen for the illustration. We thank Dr. Eugene B. Small of the University of Maryland for collecting the sample and depositing the strain at the ATCC.
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Key words: Ultrastructure - Taxonomy - Flagellate - Amastigomonas - Apusomonas Francis I. Molina, American Type Culture Collection, 12301 Parklawn Drive, Rockville 20852, MD, USA