An Ultrastructural Study of Lentomonas applanatum (Preisig) N. G. (Euglenida)

J. Euk. Microbid., 41(2), 1994, pp. 112-1 19 0 1994 by the Society of Protozoologists

An Ultrastructural Study of Lentomonas applanatum (Preisig) N. G . (Euglenida) MARK A. FARMER*‘ and RICHARD E. TRIEMER** *Centerfor Advanced L‘ltrastructural Research, Barrow Hall, University of Georgia, Athens, Georgia 30602 and **Department of Biological Sciences and Bureau of Biological Research, Rutgers University, Piscataway, New Jersey 08855-1059

ABSTRACT. Lentomonas applanatum (syn. Entosiphon applanatum Preisig) is a biflagellate, phagotrophic euglenid found in intertidal salt marshes. Lentomonas applanatum bears a superficial similarity to Entosiphon sulcaturn, however, an ultrastructural study of L. applanatum revealed many features that are atypical for other described species of the genus Entosiphon. These features include number and organization of pellicular strips, construction of the feeding apparatus, nature of the flagellar transition zone and flagellar apparatus, and point of flagellar emergence. These differences show that L. upplanaturn is related more closely to phagotrophic genera such as Ploeotia than to E. sulcatum. The construction of the feeding apparatus and pellicle suggest that L. applanatum has retained many of the pleisiomorphic characters that were present in the earliest euglenids. The presence of similar structures in other related protists may provide important clues as to the evolution of the Euglenida. Supplementary key words. Entosiphon, euglenid, evolution, Lentomonas, taxonomy, ultrastructure.

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N his original description of Entosiphon, Stein [20] cited several features as characteristic to the genus. The first was that cells were heterodynamically biflagellate, with one flagellum being recurrent while the other is directed anteriorly. Another distinguishing feature was the prominent feeding apparatus that was easily visible with light microscopy. This feeding apparatus or “siphon” underwent repeated extensions and retractions from the cell body. A third feature was the presence of a rigid pellicle composed of 10 prominent strips that extended the length of the cell, longitudinally arranged, and do not form a helix. These features continue to be the criteria by which euglenid species are assigned to the genus Entosiphon [9]; to date at least 30 different species of Entosiphon have been described [7, 15, 17, 181. The discovery of a marine euglenid that exactly matches the description given for E. upplanaturn Preisig [ 151 has allowed us to compare its ultrastructure with the type species for the genus, E. sulcutum Stein. It now seems clear that the fine structure of this organism differs significantly from that of E. sulcufum, and that these differences warrant the erection of a new euglenid genus, Lentomonus. MATERIALS AND METHODS Cells ofL. applanatum were collected from field samples from an intertidal salt marsh (21 ppt) at Tuckerton, New Jersey. Living cells were examined in a Zeiss Photomicroscope equipped with Nomarski Interference optics. Samples for scanning and transmission electron microscopy were fixed in 1.5% (v/v) glutaraldehyde, 0.5% (w/v) osmium tetroxide, and 0.05 M phosphate buffer, pH 6.8, in seawater for 10 min at room temperature. Cells then were dehydrated in a graded ethanol series. Cells used in scanning electron microscopy (SEM) were criticalpoint dried in liquid CO, and sputter-coated with Au-Pd prior to viewing in a Hitachi S450 or Philips 5 0 5 SEM. Samples for transmission electron microscopy (TEM) were transferred to acetone following ethanol dehydration, and subsequently infiltrated with epoxy resin and flat-embedded between Teflon-coated glass slides and cover slips. Following polymerization at 60” C, individual cells were affixed to epoxy blocks and serially sectioned. Silver-gold sections were stained with uranyl acetate pnor to viewing in either a Siemens 1A or Philips 300 TEM. RESULTS Cells of L. upplanaturn are rigid. ventrally flattened, and oval when viewed from the dorsal side (Fig. 1-3). The pellicle is

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To whom correspondence should be addressed.

composed of 10 longitudinally arranged ridges (seven on the dorsal side, three on the ventral surface), two of which form a channel on the ventral surface in which the posterior flagellum lies (Fig. 1-3, 5). The anterior flagellum is slightly less than body length and beats with an undulating motion while the thicker posterior flagellum trails underneath the cell’s ventral side and is one and a half to two times body length (Fig. I). A large nucleus with permanently condensed chromosomes and a single nucleolus lies to the cell’s left (when viewed dorsally) in a median to anterior position (Fig. 5, 12, 18). Numerous mitochondria with plate-like cristae are present throughout the cytoplasm (Fig. 5 , 17) as are several Golgi apparatuses, including one that apparently produces the feeding apparatus vesicles (Fig. 12). Trichocysts were seen near the periphery of the cell and are docked beneath the crests of pellicular ridges (Fig. 11). Food vacuoles were primarily found in the posterior portion of the cell (Fig. 18). Paramylon grains were not observed. Feeding apparatus. The feeding apparatus of L. upplanaturn i s composed of two supporting rods and four associated central vanes (Fig. 5, 15, 16). Originating in the most posterior portion of the cell, the feeding apparatus extends the entire length of the cell and lies adjacent to the ventral surface (Fig. 5 , 13-17). At its distal end, the feeding apparatus appears as a single row of microtubules adjacent to the pellicle (Fig. 17). Anterior to this region, a number of microtubules are added to the original row (Fig. 13). These microtubules are arranged into a W-shaped configuration and have four vanes closely appressed to them (Fig. 13). The central vanes extend the length of the feeding apparatus (Fig. 18); each terminates on a single microtubule (Fig. 13-16). An electron-dense material is associated with the outermost microtubules (Fig. 13, 14). Eventually this material becomes organized into the two supporting rods of the feeding apparatus and a small group of microtubules becomes associated with the central portion of each rod (Fig. 15, 16). One supporting rod has 10 to 12 central microtubules arranged in a single row while the other rod has its central microtubules arranged in a matrix (Fig. 15, 16). The microtubules, which are continuous at the posterior end of the feeding apparatus, (Fig. 13) separate anteriorly and eventually line the inner sides of the supporting rods (Fig. 15, 16). As they proceed anteriorly, the four vanes change from their folded or “plicate” arrangement (Fig. 13, 14) and move away from the supporting rods into a more central position (Fig. 15, 16). A secondary thickening, associated with each vane, is visible mid-way along the feeding apparatus and becomes more prominent toward the anterior end as the vanes themselves become smaller (Fig. 4). At the posterior end of the feeding apparatus, the two supporting rods are united by an electron-dense bridge

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and the microtubules associated with the outer and central portions of the supporting rods terminate (Fig. 4). Several other structures are also present in the anterior portion of the feeding apparatus. One of these, the anterior cap, is composed of electron-dense material embedded in the cytoplasm. A large bundle of microtubules arises from the anterior cap and forms a ring that partially encircles the feeding apparatus (Fig. 4). This ring of microtubules extends to the side of the flagellar opening away from the feeding apparatus, where it appears as a microtubular comb (Fig. 4). The actual cytostome is surrounded by the central vanes and their thickenings (Fig. 4). A large number of vesicles are associated with the anterior portion of the feeding apparatus (Fig. 4, 12, 18). In addition to the microtubules of the supporting rods and comb, there is a small group of microtubules that extends from the flagellar opening toward the cytostome (Fig. 4, arrowhead). N o pocket or cavity was found associated with these microtubules. Examination of living cells suggests that the feeding apparatus of L. applanatum does not protrude from the cell. Pellicle. The pellicle of L. applanatum is composed of 10 prominent ridges that extend longitudinally along the axis of the cell (Fig. 1-3). Cells are rigid and were never observed to undergo metaboly. The outermost edge of each pellicular ridge is unevenly bifurcate (Fig. 5). Microtubules underlie the crest of each pellicular ridge (Fig. 14) but were not observed in the region between pellicular ridges. All the pellicular ridges are united at the cell’s posterior end and form a slight depression at the point of their union (Fig. 1, 3, 17). At the anterior end of the cell, the pellicular ridges curve over and extend down the ventral surface (Fig. 1, 2). The ridges become smaller as they proceed along the ventral groove until eventually their microtubules become associated with those of the flagellar opening (Fig. 4). Flagellar apparatus. The two flagella ofL. applanatum emerge from an opening on the ventral surface of the cell. The thicker of the two flagella is directed posteriorly, adjacent to the cell’s ventral surface, and is designated as the posterior flagellum while the thinner anterior flagellum is directed anteriorly (Fig. 1). Both flagella have paraxial rods, with the paraxial rod ofthe posterior flagellum being more prominent and having an electron-dense structure associated with it (Fig. 7, 10). Sudden contractions of the posterior flagellum result in a change of direction for the cell, but usually the posterior flagellum is passively dragged along the substrate. In contrast the anterior flagellum actively beats along its entire length. The flagellar apparatus of L. applanatum consists of two functional basal bodies, microtubular roots, and several connective fibers. The transition zone between the axoneme and basal body is characterized by an electron-dense constriction (Fig. 6-8). Beneath this region, a central structure extends the length of the basal body and gives it a nine-plus-one pattern (Fig. 9a-c). The basal bodies occupy a ribosome-free zone at the base of the reservoir. A striated connective fiber extends between the two basal bodies and attaches to some of the triplets of each basal body (Fig. 9b, c). When seen in longitudinal section, the connective fiber did not appear as an extensive sheath that ran the length of the basal bodies but rather as a relatively fine fiber connecting the basal bodies. In addition to the striated connective fiber, a thin fiber connects the posterior basal body to the pellicle (Fig. 7, 8). A similar fiber connecting the anterior basal body to the pellicle was not observed. Whereas several distinct groups of microtubules can be seen surrounding the flagellar reservoir (Fig. lo), only the intermediate root can be clearly observed in the basal body region of the cell. The seven-member intermediate root (Fig. 9a) originates adjacent to the posterior basal body and proceeds ante-

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1 Fig. 1. Diagrammatic representation of L. upplanaturn in dorsal (a) and ventral (b) view.

riorly between the two basal bodies. Serial sectioning did not reveal additional basal bodies in interphase cells. DISCUSSION The general ultrastructure of L. applanatum is similar to that described for other colorless euglenids [4, 11, 13, 23, 25, 261. Trichocysts of the type found in L. applanatum have also been reported in Entosiphon sulcatum [ 1 1, 121 and could be interpreted as an indication that the two species are congeneric. The fact that similar trichocysts have been observed in some bodonids [2], the phagotrophic euglenids Anisonema [25] Dinema (MAF, unpubl.), and other related protists [ 161 suggests that the presence of trichocysts in L. applanatum represents a pleisiomorphic condition. Despite its superficial similarity to E . sulcatum, L. applanatum has none of the specific characters that would link it with E. sulcatum. There are, however, a number of characters that distinguish L. applanatum from all other colorless euglenids. With two functional basal bodies, a striated connecting fiber, and microtubular roots, the flagellar apparatus of L. applanatum is similar to those described for many euglenids [5]. As in all other euglenids that are heterodynamically biflagellate, the functional basal bodies are connected by way o f a prominent striated connecting fiber [5, 6, 8, 191. Despite these similarities the flagellar apparatus of L. applanatum differs from those described for other euglenids in two ways. First, the flagellar transition zone has a large, opaque, constricted region that is unlike any in other euglenids [5, 6, 8, 191. The small plate in the transition zone of E. sulcatum [8] is quite different from the type of transition zone found in L. applanatum. Second, the lack of additional basal bodies in interphase cells has only been reported for certain members of the Euglenales [ 14, 2 1, 28, 291, whereas all the phagotrophic species that have been examined, including E . sulcatum, possess additional basal bodies during the majority of interphase [3, 5, 6, 8, 19, 261.

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Fig. 2-4. L. applanaturn. 2. General cell shape whcn viewed dorsally. Bar = 2.7 pm. 3. View showing pronounced flattening of cell. Arrowhead denotes pellicular bifurcation. Bar = 1.7 prn. 4. Transverse section through anterior portion of the feeding apparatus showing relative positions of united supporting rods (SR), feeding comb (Cb), vanes (V), vesicles (Ve), anterior cap (AC), remains of flagellar opening (asterisk) and additional group of rnicrotubules (arrowhead). Bar = 0.5 prn.

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Fig. 543. Electron micrographs of L. applanatum. 5 . Median transverse section showing r as the nucleus (N), mitochondria (M), feeding apparatus (FA), anterior (AF) and posterior (PF) flagella as well as three ventral pellicular ridges (#1-3) and seven dorsal ridges (#&lo), each of which is bifurcate (arrowhead). Ventral surface is oriented towards bottom of micrograph. Bar = 1.0 pm. 6. Longitudinal section through the anterior flagellum (AF) showing electron-dense constriction in transition zone between flagellum and basal body (arrowhead). Bar = 0.5 pm. Fig. 7, 8. Adjacent longitudinal sections through posterior flagellum basal region. 7. Ventral flagellum with prominent paraxial rod (PAR), transition zone (arrowhead), and connective fiber (arrow). Bar = 0.5 pm. 8. Connective fiber (arrows) attaches the posterior basal body to the pellicle. Bar = 0.5 pm.

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Fig. 9-12. L. upplanaturn. 9a-c:.Non-adjacent series through flagellar bases arranged anterior to posterior showing relative positions ofanterior (AB) and posterior (PB) basal bodies, striated connective fiber (SCF), and intermediate root (IR). Bar = 0.5 gn. 10. Transverse view of the flagellar reservoir with surrounding microtubules (arrows), anterior flagellum (AF), and posterior flagellum (PF) with its well developed paraxial rod (PAR) and electron-dense inclusion (arrowhead). Bar = 0.5 pm. 11. Tangential section of bifurcate pellicular ridge with docked trichocyst (T). Scale = 1.0 Fm. 12. Tangential section through anterior portion of the cell showing a large Golgi apparatus (G) with associated vesicles (Ve) and various components of the feeding apparatus. Bar = 1.O pm.

Fig. 13-18. Feeding apparatus. 13-16. Nonadjacent transverse sections through the feeding apparatus arranged from posterior of cell to anterior. 13. Feeding apparatus in posterior of cell at which point microtubules appear as a single row with four associated vanes. Additional microtubules can be seen lining portions of the row. Each vane is associated with a terminal microtubule (arrow). Bar = 0.5 pm. 14. Section slightly anterior in which the single row of microtubules has separated (arrowhead) and two distinct supporting rods with associated electrondense material become apparent. Arrow shows terminal microtubule of a central vane. Pellicle microtubules (Mt) are separate from those of the feeding apparatus. Bar = 0.5 pm. 15. Section midway through cell in which the two supporting rods (SR) are clearly separate; each associated with a pair of central vanes. Note 3 x 5 matrix of microtubules in one supporting rod and the single row of eleven microtubules in the central region of the other supporting rod. Arrow shows terminal microtubule of a central vane. Bar = 0.5 pm. 16. Section through anterior of the cell where the vanes have detached and migrated to a position between the supporting rods. Arrow indicates terminal microtubule of a central vane. Bar = 0.5 p m . 17. Transverse section through posterior of cell showing microtubules of the feeding apparatus (arrowhead) extending to the posterior end of the cell. Some pellicle ridges have already begun to terminate in a ventral groove (VG). Bar = 1.0 pm. 18. Tangential section showing extent of the central vanes (V), cytostome (arrowhead), and numerous vesicles (Ve) near cell's anterior as well as a large food vacuole (FV). Bar = 1.0 pm.

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The presence of food vacuoles in L. applanatum and the rum has many of the characteristics found in Entosiphon species nature of its supporting rods and vanes suggests that L. ap- ( e g prominent pellicular ridges, heterodynamic flagellation, wellplanatum is an active bacteriotroph [25]. The ability to ingest developed feeding apparatus) and based solely on light microlarger eukaryotic prey is restricted to relatively few phagotrophic scopic observations the original assignment of L. applanatum euglenids [7, 251. Lentomonas applanatum lacks the enlarged to the genus Entosiphon was appropriate [ 151. What Preisig [ 151 vestibulurn and flexible pellicle that are found in euglenids such could not determine with light microscopy was that L. applanas Peranerna, Dinema, and Anisonema that are capable of in- aturn did not share many ultrastructural features with E. sulgesting eukaryotes [25, 261. Although the feeding apparatus of catum and that a more appropriate taxon was required. L. applanatum is similar to those described for other phagoOf the many described species of Entosiphon [7, 15, 17, 181, trophic euglenids [25, 26, 301 it differs in several important only the type species E. sulcatum has been examined with elecaspects. Unlike the supporting rods of Peranetna [ 1 1, 131, A n - tron microscopy [ 1 I , 12, 19, 24, 271. While light microscopic isonema [ I I , 221, Dinema [ 5 , 25, 261, Urceolus [25], and E. observations show that L. applanatum bears a resemblance to sulcatum [ 1 1 , 25, 271, the supporting rods of L. applanatum are E. sulcaturn, a careful examination of the ultrastructural charcomposed of relatively few microtubules. Although biochemical acters reveals that the two species differ significantly in terms studies have not been camed out it seems likely that the elec- of their flagellar apparatus, feeding apparatus, and pellicular tron-dense material associated with the supporting rods of L. structure. Based on these features it is clear that the two organapplanatum is identical to the rnicrotubule “cement” found in isms d o not belong to the same genus and that L. applanatum other heterotrophic euglenids [ 11. The feeding apparatus of L. cannot be assigned to any existing euglenid genus. For this reaapplanatum may represent an intermediate form between that son we have chosen to erect the new genus Lentomonas to of Ploeotia and Urceolus. Two of the four vanes in the posterior describe the “leisurely manner” in which the cell moves. portion of the feeding apparatus of L. applanatum are highly Lentomonas n. g. bent, giving them a folded appearance. In this respect the vanes of L. applanatum are similar to the plicate vanes described in Diagnosis. Colorless euglenid found moving slowly among Ploeotia [ 1, 4, 23. 25, 261. substrate and/or detritus with a creeping motion. Cells are hetThe feeding apparatus of L. applanaturn differs markedly from erodynamically biflagellate with both flagella possessing paraxial the one described for E. sulcaium. In E. sulcatum the supporting rods and emerging from a subapical opening. The transition rods are composed of closely packed microtubules and are cen- region of each flagellum is constricted slightly and is occluded trally located within the cell [ 1 1 , 271, whereas in L. applanatum by a dense matrix. The thinner flagellum is directed anteriorly the rods contain few microtubules and lie adjacent to the pellicle. and beats with a fairly regular movement. The somewhat thicker In E. sulcatum one of the two supporting rods is split and and longer posterior flagellum contracts irregularly, is directed together with the other rod forms a horseshoe-shaped ring around posteriorly, and lies adjacent to the cell’s ventral surface. Cells the central vanes and microvesicles [ 1 I , 271. This massive struc- possess a feeding apparatus composed of two supporting rods ture can be actively protruded from the anterior portion of the and four vanes that extend for nearly the entire length of the cell and has the appearance of a siphon, from whence Entosiphon cell. The supporting rods are reinforced by a few central microdraws its name. The two supporting rods of L. applanaturn are tubules and a partially encircling ring of microtubules. The four not split and were not observed to protrude from the cell. Thus vanes are of approximately equal length, are centrally positioned the feeding apparatus of L. applanatum is closest in form to that between the supporting rods, and form a folded, plicate arof Ploeotia [4, 231, not E. sulcalurn. rangement at their posterior end. Cells are rigid with a pellicle With its 10 longitudinally arranged ridges, the pellicle of L. composed of longitudinally arranged ridges, each of which is applanaturn superficially resembles the one described for E. slightly bifurcate on its outermost edge. Lentomonas applanasulcatum [lo, 111 but differs in several important respects. In turn Farmer & Triemer, nov. gen. T:Tuckerton, Ocean County, E. sulcatum the cell body is radially symmetrical with the flagella New Jersey, small pool on tidal salt marsh (21 ppt.), March emerging from an apical opening. The pellicle of L. applanatum 1984. Holotype collection no. F l , M. A. Farmer, Chrysler Heris greatly flattened on its ventral surface and the two flagella barium, Rutgers University, Piscataway, New Jersey. emerge from a ventral opening that is nearly lateral in placement. Furthermore, bifurcate pellicular ridges of the type found Lentomonas applanatum n. comb. (Preisig 1979) in L. applanatum are not evident in E. sulcaturn [ 10, 111. Cells rigid, and obviously ventrally flattened, body shape oval, Other euglenids are known to have pellicles with bifurcate 1 3.5- 1 9.5 p m long, 1 6 1 4 pm wide, and 6-8 pm thick, posterior ridges [4, 23, 251. Two of these, Ploeoria costata and Ploeoria vitrea, have an additional similarity to L. applanaturn in that frequently being slightly rimmed. Also in the anterior with slight their pellicles are composed of 10 longitudinally arranged ridges rim at the spot where the vestibulum opens. Pellicle with 10 longitudinal ridges separated by many wide and rounded fur[4]. In the case of P. vitrea two of the ridges are modified on rows. Seven to eight ribs are located on the obviously convex the ventral surface just as they are in L. applanarurn [4]. Despite dorsal side, with 2-3 small ribs on the flat to slightly convex of these striking similarities, profound differences in the feeding apparatus, flagellar transition zone, and position of the flagellar concave ventral side. The anterior flagellum is usually somewhat opening suggest that L. applanaturn does not represent a new shorter than the body, while the posterior flagellum is 1.5-2.0 times the body length. Cytoplasm colorless, never rich in grains. species of Ploeotia. The nucleus is indistinct and lies slightly left of the cell’s center. The flagellar opening in L. applanatum is unlike that described for any other euglenid. While the basic structure is simACKNOWLEDGMENTS ilar to other euglenids in that microtubules of the pellicle invaginate to line the flagellar opening, the ventral displacement The authors acknowledge the Bureau of Biological Research of this opening is unique. Unlike many other phagotrophic eu- of Rutgers University, the Center for Advanced Ultrastructural glenids, there is no common vestibulum shared by both the Research at the University of Georgia, and the National Science feeding and flagellar openings. Foundation (BSR 87-00087) for their support of this research. The organism described in this paper was identified synon- The authors are also grateful to Dorset W. Trapnell for providing ymously with E. applanatum Preisig [ 151. Lentomonas applana- the illustrations.

FARMER & TRIEMER-ULTRASTRUCTURE OF LENTOMONAS APPLANATUM N . G.

LITERATURE CITED 1. Belhadri, A., Bayle, D. & Brugerolle, G . 1992. Biochemical and immunological characterization of intermicrotubular cement in the feeding apparatus of phagotrophic euglenoids: Entosiphon, Peranema, and Ploeotia. Protoplasma, 168:1 13-124. 2. Brugerolle, G. 1985. Des trichocystes chez les Bodonides, un caracttre phylogtnttique suppltmentaire entre Kinetoplastida et Euglenida. Protistologica, 21:339-348. 3. Brugerolle, G. 1992. Flagellar apparatus duplication and partition, flagellar transformation during division in Entosiphon sulcatum. BioSystems, 28:203-209. 4. Farmer, M. A. & Triemer, R. E. 1988a. A redescription of the genus Ploeotia Duj. (Euglenophyceae). Taxon. 37:3 19-325. 5 . Farmer, M. A. & Triemer, R. E. 1988b. Flagellar systems in the euglenoid flagellates. BioSystems, 21:283-292. 6. Hilenski, L. L. & Walne, P. L. 1985. Ultrastructure ofthe flagella of the colorless phagotroph Peranema trichophorum (Euglenophyceae). 11. Flagellar roots. J. Phycol., 21:125-134. 7. Huber-Pestalozzi, G. 1955. Das Phytoplankton des Susswassers; Systematik und Biologie. Euglenophycean. In; Thienemann, A. (ed.), Die Binnengewasser, E. Schweizerbart’sche, Stuttgart, 16:1-606. 8. Joyon, P. L. & Mignot, J.-P. 1969. DonnCes rkcentes sur la structure de la cinetide chez les protozoaires flagellts. Annee. Biol., 8:1-51. 9. Leedale, G. F. 1967. Euglenoid Flagellates. Prentice-Hall, Englewood Cliffs, New Jersey. 10. Mignot, J.-P. 1965. Ultrastructure des Euglhiens. I. Etude de la cuticle chez differentes esptces. Protistologica, 1:5-15. 1 1. Mignot, J.-P. 1966. Structure eg ultrastructure de quelques Eugltnomonadines. Protistologica, 2:s 1-1 17. 12. Mignot, J.-P. & Hovasse, R. 1973. Nouvelle contribution a la connaissance des trichocystes: Les organes grillagis #Entosiphon sulcatum (Flagellata, Euglenida). Protistologica, 9:373-39 1. 13. Nisbet, B. 1974. An ultrastructural study of the feeding apparatus of Peranema trichophorum. J. Protozool., 21:39-48. 14. Owens, K., Farmer, M. A. & Triemer, R. E. 1988. The flagellar apparatus of Cryptoglena pigra (Euglenophyceae). J. Phycol., 24520528. 15. Preisig, H. R. 1979. Zwei neue Vertreter der farblosen Euglenophyta. Schweiz. Z. Hydrol., 41:155-160. 16. Schuster, F. L., Goldstein, S. & Hershenov, B. 1968. Ultrastructure of a flagellate Zsonema nigricans nov. gen. sp., from a polluted marine habitat. Protistologica, 4: 141-149.

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17. Skvortzov, B. V. 1957. New and rare flagellatae from Manchuria eastern Asia. Philipp. J. Sci., 86: 139-202. 18. Skvortzov, B. V. & Noda, M. 1969. On species of genus Entosiphon Stein, Fam. Peranemaceae, Euglenaceae from Sao Paulo, Brasil. Sci. Rep. Niigata Univ.. Ser. D (Biology), 6537-92. 19. Solomon, J. A., Walne, P. L. & Kivic, P. A. 1987. Eniosiphon sulcatum (Euglenophyceae): flagellar roots of the basal body complex and reservoir region. J. Phycol., 2335-98. 20. Stein, F. R. 1878. Der Organismus der Infusionstiere. 111. Der Organismus der Flagellaten. Verlag von Wilhelm Englemann, Leipzig. 2 1. Surek, B. & Melkonian, M. 1986. A cryptic cytostome is present in Euglena. Protoplasma, 133:39-49. 22. Triemer, R. E. 1985. Ultrastructural features of mitosis in Anisonema sp. (Euglenida). J. Protozool., 3 2 6 8 3 4 9 0 . 23. Triemer, R. E. 1986. Light and electron microscopic description of a colorless euglenoid, Serpenomonas costata n.g., n.sp. J. Protozool., 33:412415. 24. Triemer, R. E. 1988. Ultrastructure of mitosis in Entosiphon sulcatum (Euglenida). J. Protozool., 39231-237. 25. Triemer, R. E. & Farmer, M. A. 1991a. Ultrastructural organization of the heterotrophic euglenids and its evolutionary implications. In: Patterson, D. J. & Larsen, J . (ed.), The Biology of Free-living Heterotrophic Flagellates. Clarendon Press, Oxford. 45: 186-203. 26. Triemer, R. E. & Farmer, M. A. 1991b. An ultrastructural comparison of the mitotic apparatus, feeding apparatus, flagellar apparatus, and cytoskeleton in euglenoids and kinetoplastids. Protoplasma, 164:91-104. 27. Triemer, R. E. & Fritz, L. 1987. Structure and operation ofthe feeding apparatus in a colorless Euglenoid, Entosiphon sulcutum. J. Protozool., 34:39-47. 28. Willey, R. L. & Wibel, R. G . 1985. The reservoir cytoskeleton and a possible cytostomal homologue in Colacium (Euglenophyceae). J. Phycol., 21~570-577. 29. Willey, R. L. & Wibel, R. G. 1987. Flagellar roots and the reservoir cytoskeleton of Colacium libellae (Euglenophyceae). J. Phycol., 23:283-288. 30. Willey, R. L., Walne, P. L. & Kivic, P. 1988. Phagotrophy and the origins of the euglenoid flagellates. CRC Crit. Rev. Plant Sci., 7: 303-340.

Received 2-27-91, 8-26-93; accepted 9-02-93.

J. Euk. Microbiol.. 41(2), 1994, pp. 119-123 0 1994 by the Society of Protozoologists

Aminopeptidases from Plasmodium falciparum, Plasmodium chabaudi chabaudi and Plasmodium berghei G . PAUL CURLEY, SUSAN M. O’DONOVAN, JOHN MCNALLY, MARGARET MULLALLY, HELEN O’HARA, ALICE TROY, SUE-ANN O’CALLAGHAN and JOHN P. DALTON1 School of Biological Sciences, Dublin City University, Glasnevin, Dublin 9, Republic of Ireland ABSTRACT. Using fluorogenic substrates and polyacrylamide gels we detected in cell-free extracts of Plasmodium falciparum, Plasmodium chabaudi chabaudi and Plasmodium berghei only a single aminopeptidase. A comparative study of the aminopeptidase activity in each extract revealed that the enzymes have similar specificities and kinetics, a near-neutral pH optima of 7.2 and are moderately therrnophilic. Each has an apparent molecular weight of 80,000 ? 10,000, determined by high performance liquid chromatography on a calibrated SWSOO column. Whilst the P. c. chabaudi and P. berghei activity co-migrate in native polyacrylamide gels, that of P. falciparum migrates more slowly. The three enzymes can be selectively inhibited by ortho-phenanthroline and are thus metalloaminopeptidases; however, in contrast to other aminopeptidases the metal co-factor does not appear to be ZnZ+. Supplementary key words. Malaria, metallopeptidases, aminopeptidases.

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ROTEOLYTIC e n z y m e s play a crucial role in a wide variety of processes d u r in g the asexual erythrocytic cycle of the malaria parasite. For example, serine, and possibly cysteine

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To whom correspondence should be addressed.

proteinases are required for erythrocyte rupture and subsequent re-invasion b y merozoites [4,7, lo]. There is also extensive evidence that malaria-encoded proteinases are involved in the degradation of hemoglobin, t h e source of most of t he a m i n o acids required by t h e parasite for protein synthesis and growth [ 14, 18, 201. A number of hemoglobin-degrading proteinases