Phylogeny of Diplonema ambulator (Larsen and Patterson)

Europ. J. Protisto!' 32, 64-76 (1996) February 23, 1996

European Journal of

PROTISTOLOGY

Phylogeny of Diplonema ambulator (Larsen and Patterson) 2. Homologies of the Feeding Apparatus Ann E. Montegut-Felkner and Richard E. Triemer Department of Biological Sciences and Bureau of Biological Research, Rutgers University, Piscataway, NJ, USA

SUMMARY The ultrastructure of the feeding apparatus in Diplonema ambulator was determined and compared to other euglenozoan feeding apparatuses. A cytostome opens to the right of the flagellar pocket and continues proximally into the cell as a tubular cytopharynx. Paralleling the cytopharynx on the side facing the flagellar pocket are five vanes and a cytopharynx-associated matrix (CAM). A complex electron-dense structure surrounds the cytostome/cytopharynx, vanes, and CAM. An extension of this structure gives rise to a large band of microtubules that parallels the cytopharynx. The structure divides into a series of thin matrices which are supported by three microtubular bundles. The feeding apparatus is connected to the flagellar pocket by an anterior cytoplasmic loop that is supported by several microtubules, some of which are embedded in a dense matrix and eventually develop into vanes. On the flagellar pocket side, the microtubules are joined by another band of microtubules and continue to line the flagellar pocket as it extends into the cell. These results suggest that the feeding apparatus and flagellar pocket extension in Diplonema ambulator may represent a single, highly modified bodonid-like feeding apparatus, contradicting an earlier idea that Diplonema has two separate feeding apparatuses.

Introduction Diplonema (= Isonema) is an unusual heterotrophic protist that shares several morphological characters with members of the Euglenozoa [12, 14, 16, 18,21, 22,26]. However, the presence or absence of additional characters that are unique to the genus make speculation of its exact placement problematical. Although it is generally agreed that Diplonema probably diverged near the base of the euglenozoan lineage, it is unclear as to whether it was derived from a euglenid ancestor [18], a bodonid ancestor [12], or the ancestor that gave rise to the group as a whole [22]. The ultrastructure of the feeding apparatus has been cited as a useful character in discerning the phylogenetic relationships among the Euglenozoa [21]. Although a feeding apparatus has been reported for several species of Diplonema [16, 18, 23], the data 0932-4739-96-0032-0064$3.50-0

are incomplete making it difficult to confidently suggest structural homologies that may exist between it and the feeding apparatus found in other genera. As such, the purpose of this paper was to determine the three-dimensional structure of the feeding apparatus in Diplonema ambulator and compare it to the structure of the feeding apparatus in the bodonids and euglenids. Material and Methods Cultures and Electron Microscopy Culture maintenance and electron microscopy fixation of Diplonema ambulator followed that described previously [14]. Briefly, cells were concentrated onto Millipore filters, fixed with 2% glutaraldehyde in phosphate buffer, washed, then fixed with 2 % osmium tetroxide in phosphate buffer. © 1996 by Gustav Fischer Verlag, Stuttgart

Diplonema ambulator Feeding Apparatus . 65 For scanning electron microscopy (SEM), cells on filters were dehydrated in an ethanol series, critical point dried, coated with gold/palladium, then viewed with an Hitachi S450 SEM. For transmission electron microscopy (TEM), cells on filters were embedded in agar, dehydrated in a graded ethanol series then infiltrated with resin and polymerized. Samples were serially sectioned, floated onto formvar-coated slot grids, and post-stained with uranyl acetate and lead citrate. Sections were viewed with a lEOL 100CX II TEM equipped with a goniometer stage.

Indirect Immunofluorescence Diplonema ambulator cells were collected by centrifugation and fixed with 1.5% formaldehyde in 0.1 M phosphate buffer (pH 7.2) for 30 minutes. Fixed cells were pelleted, washed in 0.1 M phosphate buffer (pH 7.2) for 5 minutes, then transferred to a microfuge tube. Cells were then incubated in extraction buffer (0.1 % Triton X-1 00, 0.1 M phosphate buffer, 0.12% sodium azide) for 2 hours at room temperature, washed 4 times in phosphate buffer (pH 7.2) for 1 hour each, and incubated in primary antibody (rabbit anti-tubulin, Sigma catalogue number T-3526) at 4 cc for 16 hours. Cells were again washed 4 times in 0.1 M phosphate buffer (pH 7.2) for 1 hour each then incubated in secondary antibody (goat anti-rabbit FITC, Sigma catalogue number F-0382) at 4°C for 20 hours and washed in phosphate buffer (pH 7.2) for 1 hour with 15 minute changes. Microscope slides were coated with poly-I-lysine. A drop of cells was added to a coated slide and allowed to dry. Then, a drop of 90% glycerol in phosphate buffer and 100 mg/ml DABCO (1,4 diazobicyclo-[2,2,2]octane, Sigma catalogue number D-2522) was added to the cells to prevent quenching. Finally, a cover slip was placed over the cell preparation and the edges were sealed with fingernail polish. Cells were viewed with a Zeiss universal microscope equipped with epifluorescence optics.

Results

General Orientation Diplonema ambulator has two flagella that insert subapically into a flagellar pocket (Figs. 1, 4, 26). Although this organism undergoes drastic changes in cell shape (Figs. 2, 3), the flagellar pocket always lies to the left of the complex feeding apparatus (= type II feeding apparatus of [21]). However, because of the changes in cell shape, the exact angle and displacement between the flagellar pocket and the feeding apparatus are not constant. As such, the plane of section through the flagellar pocket may not be the same in similar planes of section through the feeding apparatus. For the ease of presentation, the cross-sectional views of the feeding apparatus are presented in the same orientation with respect to the drawing (Fig. 26). The arrows (lower right corner, Fig. 26) indicate relative directions with respect to spatial orientation.

Anterior Connective Loop and Parallel Microtubule Loop The flagellar pocket and the feeding apparatus are connected by a cytoplasmic extension (AC, Fig. 1) that is supported by the anterior loop (AL, Fig. 4) and parallel microtubule loop (PML, Figs. 7- 8). A fibrous matrix is sometimes seen beneath these microtubular bands (arrowheads, Fig. 5). The AL consists of 4 microtubules each embedded in a dense matrix (Figs. 7,9,10, 23, 26). It is continuous on one side with 4 of the matrix-surrounded microtubules of the microtubuleassociated region of the flagellar pocket (= MTR or type I feeding apparatus of [21]; see below for description). The other end of the AL is continuous with the matrix-surrounded microtubules that give rise to the vanes of the feeding apparatus (see Feeding Apparatus section). Below the anterior connective, the PML is composed of 3-4 microtubules and parallels the AL (Figs. 7-8). Like the AL, this microtubular band also descends along the flagellar pocket and feeding apparatus as it enters the cell (Figs. 12, 19, 26).

Microtubule-Associated Extension of Flagellar Pocket (Pocket Extension) The pocket extension originates as the right-ventral side of the flagellar pocket [14]. This structure extends below the flagellar pocket and makes a twisting J-like turn as it extends proximally in the cell (Fig. 4; region 3, Fig. 26). As such, the pocket extension can appear in both longitudinal and cross-section (Fig. 4) or it can be seen in mirror-image cross-sections (Fig. 10) within a single micrograph. A zone of exclusion surrounds the cytoplasmic side of the pocket extension (Figs. 4, 17 -18). The associated microtubules are derived from three components, the AL, the PML, and the external microtubule band (EMB). The components of the AL and PML can be seen in oblique cross-section as they begin to turn and descend along the flagellar pocket (Figs. 6-8). In a tilted section (Fig. 9) of this region of the cell, one can discern the 4 matrix surrounded microtubules derived from the AL, the unorganized microtubule bundle derived in part from the PML, and the microtubules derived from the EMB. The matrix surrounding the AL microtubules becomes less distinct as the pocket extension continues proximally in the cell (compare Figs. 9 and 10). Proximal to the anterior connective, additional microtubules emerge and parallel the PML, producing an unorganized bundle of 5 to 9 microtubules (Figs. 9, 10). At the cell anterior, the microtubules of the EMB approach the end of the peripheral microtubule band (PMB) of the feeding apparatus, but do not appear to merge with these microtubules. The microtubules of the EMB gradually become embedded in a dense matrix (region 2, arrowheads, Fig. 26) as they line the distal region of the pocket extension. Proximally, the matrix surrounding the microtubules resembles that surrounding the microtubules derived from the AL (Figs. 10, 15). The EMB and

66 . A. E. Montegut-Felkner and R. E. Triemer

Dip/onema ambu/ator Feeding Apparatus . 67

PML microtubules gradually terminate near the proximal end of the pocket extension (region 3, Fig. 26).

Feeding Apparatus The complex feeding apparatus of Diplonema ambulator is composed of 9 elements: the cytostome/cytopharynx, the AL-derived matrix and microtubules, the vanes, the PMB with associated matrix, the PML-derived microtubules, the cytopharynx-associated matrix (CAM) and microtubules, three microtubule bundles (a-c) with associated matrix, a fibrous connective, and the encircling microtubules (EM) (Fig. 26). It descends approximately one third the distance into the cell then makes a twisting J-Iike turn (arrowheads, Figs. 2-3,24-25). The feeding apparatus may make several turns in the cell in the proximal region and therefore may be represented by several profiles within a single micrograph. The feeding apparatus is surrounded by endoplasmic reticulum (ER, Figs. 12-14, 16-25). The cytostome (Cs, Figs. 8,24,26) opens to the cell exterior, lies in the right region of the feeding apparatus, and is surrounded by the majority of other components. As the feeding apparatus descends into the cell, the cytostome continues as a tubular cytopharynx that tapers proximally (compare Figs. 11-14 with Figs. 2021; region 1 with region 9 ofFig. 26). Many vesicles are seen surrounding the feeding apparatus and are believed to be partially derived from the cytopharynx (Figs. 16-19, 23). At the proximal end of the feeding apparatus, the cytopharynx is often enlarged and convoluted (Fig. 22), sometimes terminating in a large vacuole (data not shown). The components of the AL are seen in oblique crosssection as they begin to turn and descend along the feeding apparatus adjacent to the cytopharynx (Figs. 6-8, 23-24). As the AL enters the cytoplasm on the cytostome side, the surrounding matrix takes the form of 4 slingshot-like structures in cross-section

~

(Fig. 11). The "handles" of the sling shot-like structures soon disappear as the vanes become apparent and the 4 embedded microtubules protrude between the arms of each slingshot (Fig. 12; region 1, m, Fig. 26). At this point, the vanes begin to emanate toward the cytopharynx from each arm of the slingshots. The result is 5 vanes: the two terminal vanes each consist of a straight, thin lamella and the three central vanes are folded lamellae (Figs. 12-14, 16, 19, 21, 24-26). The folded nature of the central vanes arises from the lamella partially emanating from adjacent slingshot arms (i.e., the upper folded vane arises from adjacent arms of the first and second slingshot, the central vane arises from adjacent arms of the second and third slingshot, and the third folded vane arises from adjacent arms of the third and fourth slingshot) (Fig. 12; region 4, Fig. 26). As the feeding apparatus descends into the cell, the vanes continue to elongate toward the cytopharynx (Figs. 12-14, 26). They remain connected by the slingshot-derived matrix derived associated with the 4 AL-derived microtubules (Figs. 12-14, 19; regions 5, 6, Fig. 26). At the proximal extreme, the vanes become highly reduced (Fig. 22). The PMB is a semi-circular band of longitudinally oriented microtubules which surrounds the outer edge of the feeding apparatus (Figs. 23 - 26). At its distal extreme, the end which is not associated with the other complex structures of the feeding apparatus always lies near the flagellar pocket-associated EMB (Fig. 12; region 1, Fig. 26). Approximately 70-80 matrix embedded microtubules comprise the PMB at its distal end (Figs. 11-13, 26). As the feeding apparatus descends into the cell, the matrix gradually disappears in a clockwise direction until only a small amount of matrix remains associated around the right-dorsal end of the band (small arrowhead, Figs. 11-14). Here, the matrix expands in width and remains associated with the microtubules nearly the entire distance of

Figs. 1-10. General orientation, anterior connective, and development of pocket extension. - Fig. 1. SEM of cell showing relative positions of the flagella (f), the anterior connective (AC), and the feeding apparatus (FA). Bar =lllm. - Figs. 2 and 3. Immunofluorescent micrographs illustrating the variation in cell shape. Arrowheads indicate area of the feeding apparatus where it begins to twist and form the .I-like hook. Bar = 20 Ilm. - Fig. 4. Median longitudinal section through cell illustrating the relative positions of the flagellar pocket (P), the anterior loop (AL), the feeding apparatus (FA), and the pocket extension (PE). Note the twisting of the PE, resulting in a cross-sectional profile distal to the proximal curve. f = flagella. Bar =lllm. - Fig. 5. Oblique median section through the anterior connective (AC) showing the presence of fibrous material (arrowheads) which lies between the subtending microtubules and the cell body. FA = feeding apparatus. Bar = 0.5 Ilm. Figs. 6-8. Non-adjacent serial sections showing the connection of the feeding apparatus and the flagellar pocket. At the distal end, the anterior loop (AL) is seen in oblique longitudinal secrion (Fig. 6). As the series continues proximally through the cell, the AL appears as two oblique cross-sectional profiles as one end bends towards the flagellar pocket (a) and the other end bends towards the feeding appilratus (b) (Figs. 7 alld 8). The parallcllllicroruoulc loop (PM!.) and the peripheral microtnoule hundle (PMB) are also visible in oblique section. Cs =cytostome. Bar =O.S Ilm. - Fig. 9. Cross-section through the flagellar pocket at its distal end. Note the four matrix-embedded microtubules derived from the anterior loop (AL), the cross-section of microtubules derived in part from the parallel microtubule loop (PMl), and the oblique section through the microtubules of the external microtubule bundle (EMB). Bar = 0.5 pm. - Fig. 10. Cross-section through the pocket extension above the proximal curve. The profile at the top of the micrograph is distal to the one at the borrom (small arrow). EMB = microtubule and matrix derived from the external microtubule bundle, A!. =microtubulcs and matrix derived from the anterior loop, PML = microtubules derived in part from the parallel microtuhule loop. Bar = O.S Ilm.

68 . A. E. Montegut-Felkner and R. E. Triemer

Diplonema ambulator Feeding Apparatus . 69 the feeding apparatus (small arrow, Figs. 16-21; region 4, large arrow, Fig. 26). The number of microtubuIes in the PMB also gradually decreases in a clockwise direction as the apparatus descends into the cell (Figs. 16-21). Near the proximal end, the microtubules separate into 2 bands (Figs. 20 - 21). One band remains associated with the expanded matrix while the other band becomes associated with the microtubules derived from the PML. The PML is composed of 3 -4 microtubules as it descends into the cytoplasm next to the AL and adjacent to the cytostome (Fig. 26). It continues to run adjacent to the cytopharynx, apparently to the proximal extreme of the feeding apparatus (Figs. 12, 15, 18-20; regions 5, 6, 9, Fig. 26). It becomes associated with the PMB after the latter band of microtubules splits into 2 bands (see above). The CAM is a complex structure composed of a dense matrix with 3-4 embedded microtubules (Fig. 26). At its distal end, this matrix appears amorphous to rectangular in cross-section (large arrowhead, Fig. 11). As it descends into the cell, a dense semi-circular extension wraps around the cytopharynx in a clockwise direction. Proximal to this, another dense

semicircular extension arises and is directed counterclockwise, lining the microtubules of the PMB (large arrowhead, Figs. 12-14). The CAM decreases in complexity as the feeding apparatus descends further into the cell, appearing as a simple dense matrix near the cytopharynx (large arrowhead, Figs. 15 - 21). The structure eventually terminates in the proximal region of the feeding apparatus. A large complex matrix lies between the flagellar pocket and the cytostome/cytopharynx at the distal end of the feeding apparatus (small arrow, Fig. 11; region 1, large arrow, Fig. 26). At this level, the matrix is continuous with the PMB matrix and a thickening that partially surrounds the cytostome membrane (Figs. 11, 26). A short distance below this, the matrix becomes thinner and divides into several components (small arrow, Fig. 12; region 4, Fig. 26). The thinner matrices become associated with three separate groups of microtubules which will hereon be referred to as bundles a, b, and c (Figs. 13 -14). Bundle a arises as a band of 7-8 microtubules (Figs. 12-14, 16; regions 4,5, Fig. 26). Proximally, this band splits into one band of 3 microtubules plus another band of 5 microtubules (Fig. 17). Three to four rows of 2 microtubules each are

.... Figs. 11-19. Cross-sectional series through the feeding apparatus. - Figs. 11-14. Non-adjacent serial sections in the distal region of the cell. On the feeding apparatus side, the anterior loop (AL) appears as four sling shot-like structures which then lose their "handles" as they develop into the five vanes (V) (Figs. 11, 12). The vanes elongate as they develop proximally in the cell (Figs. 12-14) and maintain their association with the AL-derived microtubules. The matrix associated with the peripheral microtubule band decreases in size in a clockwise direction until only a thickened region appears associated with the microtubules at the right-dorsal side of this semi-circular band (small arrowhead, Figs. 11-14). The amorphous cytopharynx-associated matrix (large arrowhead, Figs. ] 1-14) develops a clockwise-directed semi-circular extension then a counterclockwise semicircular extension, the latter of which parallels the peripheral microtubule band for a short distance. The matrix component between the cytopharynx and the flagellar pocket (small arrow, Figs. 11, 12) divides and associates with three microtubule bundles (a-e, Figs. 13-14). By the end of this series (Fig. 14), bundle a consists of approximately 8 microtubules, bundle b consists of a row of approximately 13 microtubules and begins to develop a second row of microtubules, and bundle c is visible in a 3 plus 1 arrangement. The three components that comprise the supporting microtubules of the flagellar pocket (fp, Fig. 11) are seen in oblique section. EMB =external microtubular band, PML =parallel microtubular loop, Cp =cytopharynx, ER = endoplasmic reticulum. Bars = 0.5 ~lm. - Fig. 15. Cross-section through feeding apparatus and pocket extension (PE). This section is from a different cell, slightly proximal to Fig. 14. The number of microtubules of the peripheral microtubule bundle (PMB) have decreased in number, the clockwise and counterclockwise directed extensions of the cytopharynx-associated matrix (arrowhead) have terminated, bundle a is composed of 8 microtubules, bundle b is composed of one row of 7 microtubules and a second row of 1 microtubule, bundle c still has a 3 + 1 configuration with the singlet microtubule displaced towards the parallel microtubule loop (PML). A fibrous connective (FC) is apparent between the PMB and matrix associated with bundle c, and the microtubule components of the PE are seen more clearly. EMB =external microtubule band, AL =matrix and microtubules derived from the anterior loop. Bar = 0.5 ).1m. - Figs. 16-18. Non-adjacent serial sections in a region proximal to Figs. 11-14. The microtubules of the peripheral microtubule band (PMB) gradually disappear in a clockwise direction; the surrounding matrix (small arrow) is only associated with the microtubules at the right-dorsal end of the semi-circle. The cytopharynx-associated matrix (arrowhead) is seen as a single amorphous unit. Bundle a divides producing a 3 + 5 configuration (Fig. 17). Additional rows of microtubules are added to the triplet microtubules while the quintet microtubules approach bundle b (Fig. 18). By the end of this series (Fig. 18), bundle b is composed of approximately 4 rows of 5 microtubules each, additional microtubules have associated with the triplet microtubules of bundle c, the AL-derived microtubules associated with the vanes (V) are still apparent, and the microtubules of the peripheral microtubule loop (PML) are seen in oblique section. Note the presence of vesicles (Ve). ep '" cytopharyllx, fp = flagellJr pocket distal to pocket extension (PE), ER = endoplasmic reticulum. Bars = 0.5 ).1m. - Fig. 19. Section through a Ilother cell in the region proximal to Figs. 16-18. The microtubules of the peripheral microtubule band (PMB) have further decreased in number and separated into two independent bands, one band still associated with the matrix component (small arrow) and the other band associated with the microtubules of the parallel microtubule loop (PML). The vanes (V) are still associated with the microtubules of the anterior loop. The cytopharynx-associated matrix (arrowhead) is still apparent. The quintet microtubules of bundle a are associated with the extensive bundle b. The number of microtubules of bundle c has increased although the singlet microtubule remains associated with the PML. ER = endoplasmic reticulum, Ve = vesicles. Bar = n.5 pm.

70 . A. E. Montegut-Felkner and R. E. Triemer

Figs. 20-25. Cross and longitudinal sections through the feeding apparatus. - Figs. 20-22. Non-adjacent cross-sections through the feeding apparatus near the proximal end. The peripheral microtubule band (PMB) becomes associated with the parallel microtubule loop (PML) (Fig. 20) then separates into two components, one of which remains associated with the matrix (small arrow, Fig. 21). The vanes (V) and cytopharynx-associated matrix (arrowhead) are still apparent. The quintet microtubules of bundle a remain associated with bundle b. The number of microtubules in bundle c increases as it approaches bundle b. Fig. 22 is a profile seen below the profile in Fig. 21 and represents the bottom of the feeding apparatus as it curves back up through the cytoplasm. As such, it is a mirror image of that in the other micrographs. The cytopharynx (Cp) has become extensive and the vanes have become reduced in size. Only one microtubule bundle (large arrow) and a few microtubules of the PMB remain. ER = endoplasmic reticulum. Bars = 0.5 j.lm. - Figs. 23-25. Non-adjacent longitudinal sections through the feeding apparatus and pocket extension (PE). This series proceeds dorsal to ventral through the cell. The anterior loop (AL) is seen in near cross-section (Fig. 23) before it descends towards the feeding apparatus (Fig. 24). The encircling microtubules (EM) are seen in cross-section (Fig. 23) then longitudinal section (Fig. 25) as the series continues ventrally through the feeding apparatus. The peripheral microtubule band (PMB) and vanes (V) are seen in longitudinal section. Note the twisting of the feeding apparatus near its proximal end (arrowheads). Cs =cytostome, Cp =cytopharynx, ER =endoplasmic reticulum, Ve = vesicles. Bars = 0.5 !-Lm.

Diplonema ambulator Feeding Apparatus . 71

then added to the triplet band (Figs. 18-21; region 6, Fig. 26). The quintet band gradually becomes associated with bundle b (Figs. 18-21). At its distal point of emergence, bundle b consists of a single band of 813 microtubules (Figs. 12 -13). Three to five additional rows of approximately 5 microtubules each are added to bundle b as it descends into the cell (Figs. 14,16-21; regions 5-7, Fig. 26). Finally, bundle c is composed of 4 microtubules arranged in a 3 + 1 configuration at its distal end (Figs. 13-14; region 4, Fig. 26). The singlet microtubule is displaced towards the PML as it descends into the cell (Figs. 15, 19; regions 5 and 6, Fig. 26). The triplet microtubules gradually become associated with additional microtubules resulting in a bundle of 4 - 5 rows of microtubules comprising 5 - 9 microtubules each. The bundles converge near the proximal region of the feeding apparatus as the microtubules decrease in number (Figs. 20-22; region 8, Fig. 26), resulting in a bundle of approximately 2 rows of 8 microtubules each (Fig. 22; region 9, Fig. 26). A fibrous connective (FC) is often seen between the matrix associated with bundle c and the expanded matrix associated with the PMB (Fig. 15; regions 4, 5, Fig. 26). Finally, at the distal end of the feeding apparatus, a band of approximately 12 microtubules (EM) lies to the inside of the PMB and runs perpendicular to it (Figs. 23-25; regions 1,4, Fig. 26). Discussion The three-dimensional reconstruction of the feeding apparatus of Diplonema ambulator has revealed a greater complexity of this structure than previously realized. The individual elements of the feeding apparatus in Diplonema ambulator are compared to the feeding apparatus elements described for other Diplonema species, bodonids, and euglenids. This discussion is followed by our ideas about the homologies of the feeding apparatus of Diplonema and other members of the Euglenozoa. Comparison of the Feeding Apparatus in Diplonema Species The feeding apparatuses of Diplonema ambulator, Diplonema (= lsonema) nigricans (= pharyngeal complex/pharynx, [18]), and Diplonema (= Isonema) papillatum (= ingestion apparatus, [16]) share many ultrastructural features. In all three species, the feeding apparatus is connected to the region of the flagellar pocket (= flagellar canal in Diplonema papillatum) opposite the flagellar insertion point by an anterior cytoplasmic loop that is supported by matrix-embedded microtubules. This cytoplasmic loop is most pronounced in Diplonema papillatum as the anterior papillum. On one side, the microtubules which subtend the cytoplasmic loop descend along the flagellar pocket below the point of basal body insertion into a proximal extension of the flagellar pocket. In Diplonema ambu-

lator, the cytoplasmic loop is supported by two separate groups of microtubules, the four-membered matrixembedded anterior loop (AL) and the three to fourmembered parallel microtubular loop (PML). As the cytoplasmic loop reaches the flagellar pocket, the AL and PML are joined by another group of microtubules, the external microtubule band (EMB). As these microtubules descend along the pocket extension, the EMB microtubules become embedded in matrix and additional microtubules are added to the PML, resulting in approximately eight matrix-embedded microtubules and 5 loosely arranged microtubules being associated with the pocket. Schuster et al. [18] state that the matrix-embedded microtubules of the flagellar pocket are continuous with the supporting microtubules and vanes (= ribs) of the feeding apparatus in Diplonema nigricans. Close observation of the cross-section of the cytoplasmic loop in this species (R, Fig. 4, [18]) reveals the presence of four matrix-embedded microtubuIes and a dense fiber. Structural and positional similarities suggest that the four matrix-embedded microtubules may be homologous to the AL and the dense fiber may be homologous to the PML in Diplonema ambulator. This and the possible existence of an EMB could explain the microtubular arrangement around the pocket in Figs. 2 and 5 [18]. The oblique cross-section of the cytoplasmic loop (Fig. 21, [16]) and the arrangement of microtubules around the flagellar pocket (Figs. 21, 29, [16]) suggest that a similar situation may also exist in Diplonema papillatum. Schuster et al. [18] also state that the microtubules which line the flagellar pocket and are continuous with the feeding apparatus originate at the proximal ends of the basal bodies in Diplonema nigricans. It has been shown that the microtubules of the flagellar roots in Diplonema ambulator are not continuous with the microtubules that line this region of the flagellar pocket [14] and there was no evidence of this occurring in Diplonema papillatum. The lack of basal body origin in Diplonema ambulator in conjunction with the fact that the cited evidence (Fig. Ie, [18]) does not show a continuity between the microtubules that line the flagellar pocket/feeding apparatus complex and the microtubular root arising from the basal bodies suggest that further evaluation of the path of the flagellar root(s) in Diplonema nigricans is warranted. The feeding apparatus in all three species of Diplonema is composed of a series of microtubules and fibers that surround a deep invagination of the cell membrane (= cytostome/cytopharynx of Diplonema ambulator, lumen of Diplonema nigricans [18], pharyngeal opening of Diplonema papillatum [16]). This cytostome/cytopharynx is associated with a series of dense-staining lamellar structures termed vanes (= dense filaments, fibrils, or ribs in Diplonema nigricans [18], 4-1obed partial rosette of fibrous dense material in Diplonema papillatum [16]) which arise from four slingshot-like extensions of the AL-derived matrix in Diplonema ambulator. Evidence that the vanes may arise in a similar manner in Diplonema nigricans is seen

72 . A. E. Montegut-Felkner and R. E. Triemer

Al

1

4

2

5

9

6

3

7 Distal Ventral

Lett

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Right

~

Dorsal

Proximal

26

Diplonema ambulator Feeding Apparatus . 73 in Fig. 5 [18] as the fibrous connections between the vanes (compare to Fig. 12 of this paper). Cross-sections of the anterior region of the feeding apparatus of Diplonema papillatum are not available for comparison. Surrounding and paralleling the feeding apparatus on the side of the cytostome/cytopharynx opposite the vanes in Diplonema ambulator is a semi-circular band of microtubules, the peripheral microtubular band (PMB). This band is evident in cross-sections of the feeding apparatus of Diplonema nigricans (Fig. 5, [18]) and Diplonema papillatum (Fig. 29, [16]) and probably represents the majority of the longitudinal support microtubules noted by Schuster et al. (Fig. 4, [18]). ER surrounds the feeding apparatus in Diplonema ambulator and Diplonema papillatum [16]. The alveoli that surround the feeding apparatus in Diplonema nigricans [18] may actually represent expanded regions of ER in this species. Because of the limited number of micrographs available, only a few additional comparisons can be made between the feeding apparatus of Diplonema ambulator and Diplonema nigricans [18). The presence of encircling microtubules (transverse microtubules, Fig. 4, [18]) is evident. The appearance of both longitudinal and cross-sectional profiles in a single section of the feeding apparatus (Plate I, Fig. 1, [18]) suggest that it also bends in the proximal region. Schuster et al. [18] note the presence of a large anterior vacuole (Plate I, Fig. 1). The placement and morphology of this vacuole are reminiscent of the proximal expansion of the cytopharynx in Diplonema ambulator. The crosssectional profile of the feeding apparatus in Diplonema nigricans (Fig. 5, [18)), which is flipped along the vertical axis with respect to the micrographs presented in this paper, also suggests the presence of microtubules that correspond to bundles a and b and of the cytopharynx-associated matrix (CAM) with both lateral extensIOns. In summary, the generalized feeding apparatus of Diplonema comprises a cytostome/cytopharynx that is supported on one side by a series of dense, lamellar vanes and on the other side by a semi-circular PMB. Additional support of the feeding apparatus may be provided by several other microtubular (bundles a, b, and possibly c; encircling microtubules) and matrixfilled (CAM) components. The feeding apparatus is

surrounded by ER and may terminate in a large vesicle. It is connected to the flagellar pocket by an anterior cytoplasmic loop that is supported by a series of microtubules. The microtubule-supported region of the flagellar pocket extends below the point of basal body insertion. Although a complete reconstruction was not presented, the ultrastructural description of the flagellar pocket/cytopharynx in Rhynchopus coscinodiscivorus [17] indicates that it is very similar to that of Diplonema, implying that these two organisms are closely related.

Comparison of the Diplonema and Bodonid Feeding Apparatuses The MTR was first described by Brugerolle et al. [3] as the dominating microtubular fiber that is associated with several bodonid feeding apparatuses. This fiber is rooted at the base of the recurrent flagellum, ascends along the bottom of the flagellar pocket, then hooks over the cell anterior beneath the preoral crest before penetrating deeply into the cell as it parallels the cytopharynx. Brugerolle et al. [3] homologized the MTR with the supporting microtubules of the proboscis in Rhychomonas nasuta [19] and Rhynchomonas metalobita [4) as well as the preoral ridge in Cryptobia vaginalis [24] and the five to six microtubules that line the cytopharynx of certain trypanosomes like Crithidia fasiculata [2]. They concluded that the MTR probably exists in all bodonids with the number of microtubules, the surrounding dense material, and the accessory fibers being variable between genera and even between species within a single genus. As first noted by Kivic and Walne [12), the cytoplasmic connection between the flagellar pocket and the feeding apparatus in Diplonema (= Isonema) is very similar in structure and position to the preoral crest found in the bodonids. In addition, the microtubules that line this cytoplasmic connection are continuous with the microtubules which line the flagellar pocket and cytopharynx in both bodonids and Diplonema. Based on the data available for Diplonema nigricans and Diplonema papillatum, Kivic and Walne [12] stated that the cytoplasmic loop was supported by a flagellar root that follows a path similar to the MTR of bodonids. Although the microtubules do travel a similar course in Diplonema ambulator, they do not arise as

.... Fig. 26. Three-dimensional model of complex feeding apparatus, flagellar pocket (P), and pocket extension (PE). Brackets represent 9 sub-regions as explained throughout the text. Dotted lines represent cutaway views to illustrate significant changes in these regions. Arrows (lower right corner) indicate approximate spatial orientation. VF and DF = ventral and dorsal flagella, EMB =external microtubule band, EM =encirding microtubules, PML =parallel microtubule loop, AL =anterior loop, PMB = peripheral microtubule band, CAM =cyropharynx-associated matrix, V =vanes, a, b, c =microtubule bundles, FC = fibrous connective, Cs = cytostome, Cp = cytopharynx. Region 1: m = central microtubules derived from AL, 55 = sling shot-like development of AL-derived matrix, large arrow =anterior matrix. Regions 1, 4: asterisks =gradual termination of matrix around PMB microtubules. Region 2: arrowheads show development of matrix around EMB microtubules, small arrows =cutaway of PML matrix to show the presence of central microrubules. Region 4: large arrow =expansion of matrix surrounding this region of PMB. Region 9: large arrows = convergence then decrease in number in microtllbules of the bundles. Bar =1 ~m.

74 . A. E. Montegut-Felkner and R. E. Triemer

a flagellar root [14]. The number and elaborate arrangement of accessory microtubules and fibers in Diplonema ambulator is unlike that found in any bodonid feeding apparatus. However, the extension of the flagellar pocket below the point of basal body insertion in Diplonema is similar to the extension in Cryptobia intestinalis and Trypanoplasma borellii [3] and the post-flagellar pit in Cryptobia iubilans [15], Cryptobia sp. [7], and Cryptobia vaginalis [24].

Comparison of the Diplonema and Euglenid Feeding Apparatuses According to Triemer and Farmer [21, 22], Diplonema has two separate feeding apparatuses. They categorized the more complex feeding apparatus as a Type II or plicate type feeding apparatus which is defined as a cytostome that is supported by two rods and a series of plicate vanes. All of the organisms with a plicate type feeding apparatus were presumed to be bacteriotrophs. However, Ploeotia costata has since been observed ingesting yeast in a contaminated culture, indicating that this type of feeding apparatus should no longer be defined as belonging exclusively to bacteriotrophs. In parallel, the definition of the Type III feeding apparatus needs to be amended so that it does not include all phagotrophic euglenoids capable of ingesting large, eukaryotic prey. In Ploeotia, each rod of the plicate feeding apparatus consists of an electron dense cylinder (= cement-like material of [1]) that extends posteriorly into the cytoplasm and is usually hollow for part of its length [22], although the cavity within each rod can be filled with an amorphous matrix [21, 22]. A single row of microtubules is found along one side of each rod [1,22] and a few central microtubules are occasionally seen [9]. At the distal end of the feeding apparatus, the rods are united by an electron dense bridge which is underlain by a row of microtubules [9]. Partially surrounding the feeding apparatus is an electron dense feeding comb with associated microtubules [9]. Due to positional similarities, it may be that the bridge and comb [9] correspond to the anterior dome of Belhadri et al. [1], although additional studies are needed to confirm this suggestion. Four vanes (= 6 ribs of [20]) surround the cytostome opening at the cell anterior [21] and are associated with the supporting rods for most of their length except at the proximal end of the feeding apparatus where the rods and vanes merge [21, 22] and their constitutive matrix, lined by microtubules, extends around the four vanes [1]. The three-dimensional structure of the feeding apparatus in Diplonema ambulator described herein does provide additional information about the structure of the feeding apparatus and does not completely support previous statements of similarity between it and the one present in Ploeotia, stressing the necessity of using complete serial reconstructions when making comparisons. First of all, it is very difficult to recognize and precisely identify the boundaries of the supporting

rods reported previously for Diplonema [21, 22]. In their schematic of a median transverse section of a Type II feeding apparatus (Fig. 13.29b, [22]), Triemer and Farmer illustrate the rods as two distinct matrices that are separate from the vanes. They state that the two components merge at the base of the feeding apparatus to form a single complex [21, 22]. This is not the case for Diplonema ambulator. The dense structures previously interpreted to represent the two rods [21, 22] have now been shown to be the right-dorsal expansion of the PMB-associated matrix (arrows, Figs. 16-22) and the CAM. Although a precise reconstruction is not yet available for comparison, the arrangement and origin of the distal bridge and comb [9] in Ploeotia do not appear to be similar to the anterior matrix and PMB in Diplonema. Also, an anterior cytoplasmic loop does not connect the feeding apparatus with the flagellar pocket in Ploeotia nor does there seem to be any microtubules that could be homologous to the matrix-associated bundles a, b, and c in Diplonema. In fact, the distal matrix and bundles a, b, and c superficially are more similar to the cement and supporting rods that are found in Entosiphon (see [1]). Secondly, neither the arrangement nor the origin of the vanes in Diplonema ambulator resemble the situation found in Ploeotia. According to the available data, the vanes in Ploeotia appear to arise in the distal region of the feeding apparatus and surround a short cytostome/cytopharynx [9]. They develop proximally as four unusually arranged dense lamellae [1, 21, 22]. The vanes in Diplonema ambulator arise as extensions of the matrix that surrounds the microtubules of the anterior loop and are associated with one side of an extensive cytostome/cytopharynx which parallels the feeding apparatus for its entire length. The plicate arrangement of the vanes is the major factor that separates the Type II feeding apparatus from the other 3 types [21, 22]. A precise definition of "plicate" is not apparent and the superficial similarities between the vanes in Diplonema and Ploeotia may not be sufficient to classify them in the same category. A more detailed reconstruction of the feeding apparatus in Ploeotia, particularly in the distal region, is needed to further evaluate the homologies, if any, that may exist between the feeding apparatus in these two species. Triemer and Farmer [21, 22] also indicated that Diplonema has a second Type I or MTRJpocket type feeding apparatus. It is defined as a cytoplasmic pocket which opens to the cell surface adjacent to the flagellar pocket and extends approximately one half to three quarters the cell length and is reinforced by microtubuies. Within the phagotrophic euglenids, it is only found in bacteriotrophic genera and is homologous to the MTRJpocket of the kinetoplastids (see above) and the cytoplasmic pocket or cryptic cytostome found in some photosynthetic euglenids. The MTRJpocket type feeding apparatus is the sole type of feeding apparatus in the euglenids Petalomonas cantuscygni and Calycimonas sp. (= Calycimonas robusta in [22]) [21,22]. In these species, the feeding apparatus merges

Diplonema ambulator Feeding Apparatus' 75

with the flagellar pocket at the cell anterior. The microtubules extend from the point of merger and may be derived in part from one flagellar root [21]. In Petalomonas cantuscugni, the root may be the ventral root [22], although the exact point of origin is unknown [9, 10]. The origin and path of microtubules lining the pocket in Calycimonas has not been determined. The MTRJpocket has also been found in two phagotrophic euglenids, each with a second, more complex feeding apparatus [21, 22]. In Dinema, a reservoir pocket is lined by microtubules derived from the ventral root [10, 22]. AType I feeding apparatus also exists in Ploeotia [22] as an anterior invagination [91 of the reservoir [21] that is lined by microtubules that originate from a flagellar root [9], perhaps the ventral root [21]. Like these phagotrophic euglenids, the identified MTRJpocket of Diplonema [23] also exists as an invagination of the flagellar pocket. However, the present study shows that this invagination occurs at the proximal end of the pocket and it may be much more extensive than that found in Dinema and Ploeotia. Also, unlike in these other genera, the microtubules that line the invagination in Diplonema are continuous with the microtubules of the cytoplasmic loop that is continuous with the more complex feeding apparatus. In addition, these microtubules do not originate as a flagellar root, at least not in Diplonema ambulator. In summary, there appear to be many structural and positional differences which suggest that the similarities between the complex feeding apparatuses in Diplonema and Ploeotia as well as between the "MTRJpocket" of Diplonema and phagotrophic euglenids may only be superficial.

Homologies of the Feeding Apparatus In a review, Kivic and Walne [121 provided morphological evidence that suggested that Diplonema (= 1sonema) was a member of the Euglenozoa [5, 6]. Among other characters, homologies between the feeding apparatuses of these organisms were proposed and used to support their ideas. Specifically, they homologized the cytopharyngeal microtubules of Diplonema with the MTR of bodonids on the basis of pathway, origin at the basal bodies, and fibrillar connectives which attach the microtubules to the membrane and form the dense rosettes. Later, Triemer and Farmer [21, 22] suggested that Diplonema actually has two distinct feeding apparatuses. They classified the microtubule-lined extension of the flagellar pocket as a Type I or MTRJ pocket type feeding apparatus and classified the more complex feeding apparatus as a Type n or plicate feeding apparatus. They did not comment on the cytoplasmic extension that interconnects th{' two "separate" feeding apparatuses. Since the "two" feeding apparatuses reported for Diplonema ambulator have now been shown to be interconnected by microtubules as in Diplonema nigricans [18] and Diplonema papillatum [16], it is more reasonable to consider these components as a single feeding apparatus that is more similar to that found

in the bodonids rather than in the euglenids. The identified Type I feeding apparatus [21, 22] actually represents a proximal extension of the flagellar pocket like that seen in many Cryptobia species [3, 7, 15,24], Trypanoplasma borelli [3], and some species of Herpetomonas [8, 11]. The identified Type II feeding apparatus appears to represent a very elaborated bodonid cytopharynx. Recently, Belhadri et al. [1] provided support for the homology between three types of euglenid feeding apparatuses by the immunological localization of a monoclonal antibody which recognized the cement in the feeding apparatuses of Ploeotia (Type II feeding apparatus), Peranema (Type III feeding apparatus), and Entosiphon (Type IV feeding apparatus). It would be interesting to see if the same antibody recognizes the complex matrix components found in the Diplonema amhulator feeding apparatus. This might provide insight into whether the feeding apparatus in euglenoids developed to supplement the MTRJ pocket type apparatus [10, 22] or if it evolved from it. As in the bodonids, the cytostome of Diplonema ambulator exists as a permanent opening that is independent of the flagellar pocket and extends posteriorly into a tubular cytopharynx. A band of microtubules ascends along the flagellar pocket then loops over beneath a cytoplasmic loop and descends along the cytopharynx. However, this complex band of microtubuies does not originate as a flagellar root like in bodonids and the complexity of microtubules and fibers that surround the cytopharynx far surpasses that found in the feeding apparatus of any bodonid studied thus far. The fact that the ventral root does not line the feeding apparatus in Diplonema amhulator may not invalidate a possible homology between the bodonid MTR and the microtubules that support the cytostome in Diplonema. Lorn et al. [13] suggested that, although the microtubules which line some trypanosome cytostomes are independent of a flagellar root, they are homologous to the bodonid MTR. Willey et al. [26] further speculated that the parasitic lifestyle of trypanosomes may have resulted in the evolution of a reduced cytopharynx where the microtu buies no longer arise as a flagellar root. In an analogous manner, the ventral root may have originally been associated with the flagellar pocket in the ancestor to Diplonema ambulator. As the pocket invaginated below the point of basal body insertion, the ventral root may have divided into two bands. One band could have continued its path which loops around the cell anterior and ultimately becomes associated with the ingestion apparatus and the other band could have remained associated with the ventral basal body. Although the evidence is weak, it does suggest that the structure of the feeding apparatus in all species of Diplonema may not be constant. A three-dimensional reconstruction of the flagellar and feeding apparatuses in other Diplonema species could reveal a species-specific difference in the path of the ventral root and the extent of complexity of the feeding apparatus

76 . A. E. Montegut-Felkner and R. E. Triemer in a similar manner as found in the genus Cryptobia [3,

7, 15, 24, 25].

Acknowledgements This research was supported by the National Science Foundation (grant no. BSR 9020325) and the Bureau of Biological Research, Rutgers University. The authors wish to thank Carole L. Lewandowski for her technical assistance and for critically reading the manuscript. This research was submitted by A. E. Montegut-Felkner in partial fulfillment of the requirements for the Ph.D. degree, Rutgers University, New Brunswick, N.].

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Key words: Diplonema - Euglenozoa - Feeding apparatus - Isonema - Ultrastructure Ann E. Montegut-Felkner, Department of Biological Sciences, Rutgers University, P.O. Box 1059, Piscataway, N] 08855-1059, USA