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The ultrastructure of Amylovorax dehorityi comb. nov. and erection of the Amylovoracidae fam. nov. (Ciliophora: Trichostomatia)
Europ. J. Protistol. 38, 29–44 (2002) © Urban & Fischer Verlag http://www.urbanfischer.de/journals/ejp
The ultrastructure of Amylovorax dehorityi comb. nov. and erection of the Amylovoracidae fam. nov. (Ciliophora: Trichostomatia) Stephen L. Cameron* and Peter J. O’Donoghue Department of Microbiology & Parasitology, The University of Queensland, Brisbane 4072, Australia; E-mail: [email protected] Received: 18 June 2001; 15 October 2001; 14 November 2001. Accepted: 22 November 2001
The ultrastructural features of the holotrichous ciliates inhabiting macropodid maruspials were investigated to resolve their morphological similarity to other trichostome ciliates with observed differences in their small subunit rRNA gene sequences. The ultrastructure of Amylovorax dehorityi nov. comb. (formerly Dasytricha dehorityi) was determined by transmission electron microscopy. The somatic kineties are composed of monokinetids whose microtubules show a typical litostome pattern. The somatic cortex is composed of ridges which separate kinety rows, granular ectoplasm and a basal layer of hydrogenosomes lining the tela corticalis. The vestibulum is an invagination of the pellicle lined down one side with kineties (invaginated extensions of the somatic kineties); transverse tubules line the surface of the vestibulum and small nematodesmata surround it forming a cone-like network of struts. Cytoplasmic organelles include hydrogenosomes, irregularly shaped contractile vacuoles surrounded by a sparse spongioplasm, food vacuoles containing bacteria and large numbers of starch granules. This set of characteristics differs sufficiently from those of isotrichids and members of the genus Dasytricha to justify the erection of a new genus (Amylovorax) and a new family (Amylovoracidae). Dasytricha dehorityi, D. dogieli and D. mundayi are reassigned to the new genus Amylovorax and a new species A. quokka is erected. While the gross morphological similarities between Amylovorax and Dasytricha may be explained by convergent evolution, ultrastructural features indicate that these two genera have probably diverged independently from haptorian ancestors by successive reduction of the cortical and vestibular support structures. Key words: Amylovorax; Dasytricha; Haptoria; hydrogenosomes; Trichostomatia.
Introduction The endosymbiotic association of herbivorous mammals and trichostome ciliates is well established (Corliss 1979; Van Hoven et al. 1987; Dehority 1996). At present, 15 families of ciliates have been described within the litostome subclass Trichostomatia; all are endosymbiotic within herbivorous mammals including artiodactyls, perissodactyls, rodents, anthropoid apes and macropo*corresponding author
did marsupials. Previous investigations of the macropodid marsupials (kangaroos and wallabies) for the presence of endosymbiotic ciliates have revealed a diverse fauna including the macropodiniids (Dehority 1996; Cameron et al. 2001a), cycloposthiids (Cameron et al. 2000a) and holotrichous species originally described as isotrichids (Cameron et al. 2000b). The isotrichids had previously been considered to be confined to eutherian herbivores (ruminants and rodents) so their presence in 0932-4739/02/38/01-29 $ 15.00/0
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macropodid marsupials was surprising. Analysis of small subunit rRNA gene sequences from these Australian holotrichs suggested that their placement within the Isotrichidae was incorrect (Cameron et al. 2001b). Not only did the Australian species fail to group with the isotrichids from ruminants, they also appeared to form an independent lineage of trichostome ciliates entirely separate from those species associated with eutherian mammals. The molecular data suggested that the classification of the Australian holotrichs was wrong at the level of genus (several Australian species assigned to the ruminant-associated genus Dasytricha did not group with this genus), family (all Australian species failed to group with the isotrichids) and perhaps even order. Morphologically, however, the Australian holotrichs were almost indistinguishable from the isotrichids inhabiting ruminant when silver stained specimens were examined by light microscopy. The present study was therefore undertaken to investigate the ultrastructural features of a species of Australian holotrich (formerly classified as a member of the genus Dasytricha) and reconcile the disparate conclusions drawn from previous studies of gross morphology (Cameron et al. 2000b) and molecular sequences (Cameron et al. 2001b).
Materials and methods Samples were collected from 45 eastern-grey kangaroos (Macropus giganteus), 28 western-grey kangaroos (M. fuliginosus), 32 red-necked wallabies (M. rufogriseus) and 5 quokkas (Setonix brachyurus). Stomach fluid was removed either from road-killed animals or from animals culled from wild populations. Stomach liquor was squeezed from large fibrous matter and strained through a triple layer of surgical gauze to remove fine particulate matter and nematodes. The filtrate was fixed in 2 volumes of Bouin’s fluid for light microscopy or an equal volume of glutaraldehyde in 0.2 M cacodylate buffer for electron microscopy. Samples for transmission electron microscopy were washed 3 times with 0.2 M cacodylate buffer, 3 times in distilled water and stored in 70% ethanol. Ciliates were rehydrated, purified by Percoll density gradient centrifugation (column composed of equal volume layers of sample, 25%, 50%, 75% & 100%; spun at 3000 g for 30 min.) and individual ciliates harvested by micropipette. Cells were post-fixed in 4% osmium tetroxide (unbuffered), washed 3 times in Sorensen’s phosphate buffer (pH 7.2) and twice in distilled water. Samples were dehydrated in a graded series of acetone solutions and embedded in Epon resin; ciliates were pelleted within uncured resin by mild centrifugation. Semi-thin survey sections were cut with glass knives, stained with 1% toluidine blue and used to orientate sections. Ultrathin sections (70 nm and 90 nm) were cut with diamond knives, mounted on uncoated copper grids, stained
Table 1. Morphometric characterisation of the amylovoracid ciliate Amylovorax quokka sp. nov. recovered from the quokka, Setonix brachyurus. x: mean; SD: standard deviation; CV: coefficient of variation; min: minimum; max: maximum; n: number of observations. Character Body dimensions Length, L Width, W Shape index (L/W ratio) Macronucleus Length Width Micronucleus Length Width Oral apparatus Width Depth Length of cytopharyngeal rods Somatic ciliature Number of kineties (per side) Length of cilia
x
SD
CV
min
max
n
59.5 23.0 2.0
12.14 8.39 0.28
21.8 28.0 13.6
31.2 15.2 1.35
88.8 49.6 2.6
30 30 30
15.7 7.8
3.98 2.06
25.4 26.6
6.4 4.0
21.6 11.2
30 30
2.3 1.7
0.51 0.41
22.3 24.2
1.6 0.8
3.2 2.4
15 15
3.8 4.6 5.7
0.69 1.14 4.40
18.0 24.6 77.1
3.2 3.2 0.8
5.6 8.0 16.8
30 30 24
26.4 4.9
5.67 1.198
21.5 24.6
17 3.2
41 7.2
30 4.9
Ultrastructure of Amylovorax
with 5% uranyl acetate in 50% methanol for 6 min, washed in distilled water for 30 sec and dried. The sections were then counter-stained with Reynold’s lead citrate for 3 min, washed in distilled water for 30 sec. and dried prior to examination in a JEOL 1010 transmission electron microscope. Samples for scanning electron microscopy were washed with distilled water and purified by Percoll density gradient centrifugation as described above. Ciliates were washed 3 times in Sorensen’s phosphate buffer (pH 7.2), post-fixed in 4% osmium tetroxide (unbuffered), washed twice in water, dehydrated in a graded series of ethanol solutions and dried in a critical point drier between Millipore filters. Dried cells were sputtercoated with gold and examined in a JOEL 6300 scanning electron microscope. Samples for light microscopy were pelleted by mild centrifugation and washed 5–20 times with distilled water to remove Bouin’s fixative prior to storage in 70% ethanol. Ciliates were stained with silver proteinate (protargol) using a modification of Wicklow and Hill’s (1992) method and silver carbonate using a modification of the technique of Ito and Imai (1998). Ciliates were measured using a calibrated eyepiece micrometer and drawn with the aid of a camera lucida. Measurements are presented as a range of values followed by the arithmetic mean. Summary statistics of morphometric observations were prepared using the Statistix® program.
Results Holotrich ciliates detected in 23 (53%) of 43 eastern-grey kangaroos, 16 (62%) of 26 westerngrey kangaroos and 1 (3%) of 32 red-necked wallabies when examined by light microscopy were consistent with Cameron et al.’s (2000b) description of Dasyticha dehorityi. Holotrich ciliates detected in 3 (60%) of 5 quokkas differed sufficiently from the species of isotrichid-like ciliates recovered from macropodid marsupials to warrant the erection of a new species.
Ultrastructure Ultrastructure was studied in Dasytricha dehorityi, specimens of which were recovered from a single eastern-grey kangaroo (Macropus giganteus) collected at Mt. Sebastopol, Queensland, Australia. Somatic infraciliature Somatic ciliation is composed of monokinetids with a typical litostome pattern of a kinetodesmal fibril (opposite triplet 7), a convergent postciliary microtubular ribbon (opposite triplet 9) (Fig. 1A)
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and two transverse ribbons, the tangential transverse ribbon composed of 5 microtubules (T1) (opposite triplet 4) (Fig. 2A) and the radial transverse ribbon composed of a single microtubule (T2) (opposite triplet 5) (Fig. 2B). The kineties are arranged meridionally between pellicular projections termed the interkinetal ridges. The postciliary microtubular ribbon is composed of 3 microtubules which curve up from the kinetosome to run longitudinally and posteriorly through the right adjacent interkinetal ridge (Fig. 1B). The kinetodesmal fibril is of similar dimensions to the postciliary microtubular ribbon and curves up from the kinetosome to run laterally and forwards towards the n + 1 kinetosome of the adjacent right kinety (Fig. 2A). The kinetodesmal fibril passes under the postciliary ribbon of the kinetosome immediately anteriorad and is anchored in the interkinetal ridge to the immediate right. The T1 fibre curves up and anteriorly to form the right-most edge of the interkinetal ridge to the immediate left of the kinetosome. The T2 fibre is curved down and slightly anteriorly as it pierces the tela corticalis (Fig. 2B). A diagram of the three dimensional arrangement of the kineties is presented in Figure 3. Cortex The interkinetal ridges have a prominent epiplasm composed of small flattened sacs (Fig. 4A). The ridges have an apical layer of electron-dense oblong bodies placed perpendicularly to the cell membrane (Fig. 2B). These bodies vary in size from 250–300 nm but are always present as a single layer (Fig. 4B). The postciliary ribbon passes over the top of the layer separating the oblong bodies from the cell membrane (not shown). The kinetodesmal fibril passes between adjacent bodies to anchor in the apex of the interkinetal ridge (Fig. 2A). The main body of the interkinetal ridges and the region between the kinetosomes is composed of a granular ectoplasm with few inclusions (Fig. 2A). The base of the ectoplasm is made up of small ovoid hydrogenosomes which carpet the ectoplasmic surface of the tela corticalis (Fig. 4B). The tela corticalis is composed of a double layer of microfilaments which have a herringbone appearance in cross section. There is a subcortical layer composed of small, empty, membrane-bound vacuoles separating the tela corticalis from the endoplasm (Fig. 5A).
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Fig. 1. Transmission electron micrographs of A. dehorityi comb. nov. A: transverse section through somatic kinetids, scale bar = 500 nm; B: longitudinal section through somatic kinety, scale bar = 1 µm. H1: endoplasmic hydrogenosomes; H2: ectoplasmic hydrogenosomes; Kd: kinetodesmal fibril; Pc: postciliary microtubular ribbon; S: starch granule; Tc: tela corticalis.
Ultrastructure of Amylovorax
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Fig. 2. Transmission electron micrographs of A. dehorityi comb. nov. A: glancing section through somatic kineties, scale bar = 500 nm; B: glancing section through somatic kineties, scale bar = 500 nm. Db: dark body; Kd: kinetodesmal fibril; Pc: postciliary microtubular ribbon; Ps: pallicular space; T1: tangential transverse ribbon; T2: radial transverse ribbon; Tc: tela corticalis.
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with the vestibular kinetids. The vestibulum thus forms a cone which pierces the tela corticalis. There is no differentiated phagoplasm; no obvious structure separates the vestibular associated microtubules from the endoplasmic organelles.
Fig. 3. Three dimensional arrangement of cortex fibres of A. dehorityi comb. nov. Kd: kinetodesmal fibril; Pc: postciliary microtubular ribbon; T1: tangential transverse ribbon. The arrow points towards the anterior end of the cell.
Vestibulum and oral infraciliature The vestibulum is a simple conical invagination of the apical end of the cell (Fig. 5B). It is lined along one side by several kineties which are invaginated extensions of the somatic kineties. The opposite side of the vestibulum lacks cilia and is lined with evenly spaced microtubular ribbons set perpendicularly to the vestibulum wall (Fig. 5B). The vestibular kineties are separated by interkinetal ridges which are of similar structure to the somatic interkinetal ridges except that they lack the apical layer of oblong bodies (Fig. 6A). Transverse tubules associated with the bases of the kinetosomes are similar in structure to those associated with the somatic kinetosomes and are derived from the T1 microtubule (Fig. 6B). Transverse tubules also line the glabrous dorsal side of the vestibulum (Fig. 6B). The small nematodesmata are derived from an electron-dense plate at the base of the kinetosomes of the vestibular kinetids and the anterior-most kinetosomes of the somatic kinetids. The small nematodesmata are composed of 4 to 6 microtubules which pierce the tela corticalis, enter the endoplasm (Fig. 6A) and run parallel to the vestibulum, surrounding it and forming a cone-like network of struts (Fig. 7A). The tela corticalis does not line the vestibulum internally, it stops abruptly at the level of the anterior-most kinetosome of the somatic kinetids not associated
Organelles The macronucleus is surrounded by a porous nuclear envelope and contains chromatin bodies which have condensed away from the envelope (Fig. 7B). The micronucleus has a different structure; the chromatin has condensed into a single central body with a striated appearance (Fig. 7B). The endoplasm is dominated by the starch granules which make up a majority of the cell contents (Fig. 8A). There are a few food vacuoles containing bacteria. The hydrogenosomes appear to be of two types; the ectoplasm has a basal layer composed of small ovoid hydrogenosomes while the endoplasmic hydrogenosomes are larger and more irregular in shape (Fig. 4B). There is a little endoplasmic reticulum around the periphery of the endoplasm near the tela corticalis (Fig. 4A). There is a single posterior contractile vacuole which is frequently oblong or irregular in shape. There is a sparse amount of spongioplasm which does not differ greatly from the regular endoplasm (Fig. 8A). The contractile vacuole is endoplasmic; the duct pierces the tela corticalis and empties between adjacent interkinetal ridges. The duct is lined with microtubules for rigidity (Fig. 8B) and there are no accessory collecting tubules.
Taxonomy The ultrastructural features of these ciliates were sufficiently novel (cortex structure and vestibular arrangement) to justify the formal erection of a new family, as follows. Family Amylovoracidae fam. nov. Trichostomatia incertae sedis Diagnosis: Cells oval, elongate, pyriform or falcate. Somatic ciliation holotrichous with meridional kineties arranged between pellicular interkinetal ridges. Most species have prominent cytopharyngeal rods. Single macronucleus; single micronucelus generally adjacent to the macronucleus. A prominent tela corticalis divides the cell into endoplasm and ectoplasm. The ectoplasm is lined basally by small hydrogenosomes and apically by small electron-dense bodies. The kinetosome-
Ultrastructure of Amylovorax
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Fig. 4. Transmission electron micrographs of A. dehorityi comb. nov. A: glancing section through kineties, scale bar = 500 nm; B: longitudinal section through interkinetal ridge, scale bar = 500 nm. Db: dark body; H1: endoplasmic hydogenosomes; H2: ectoplasmic hydogenosomes; Ps: pallicular space; Tc: tela corticalis.
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Fig. 5. Transmission electron micrographs of A. dehorityi comb. nov. A: longitudinal section through kinety, scale bar = 500 nm; B: sagittal section through vestibulum, scale bar = 1 µm. ER: endoplasmic reticulum; Tc: tela corticalis; V: vacuole.
Ultrastructure of Amylovorax
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Fig. 6. Transmission electron micrographs of A. dehorityi comb. nov. A: oralised kineties, scale bar = 200 nm; B: sagital section though dorsal vestibulum, scale bar = 500 nm. sNd: small nematodesmata; T1: tangential transverse ribbon; TT: transverse tubules; Pl: electron-dense plate.
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Fig. 7. Transmission electron micrographs of A. dehorityi comb. nov. A: glancing section through forebody, scale bar = 2 µm; B: nuclei, scale bar = 1 µm. C: chromatin ring; Ma: macronucleus; Mi: micronucleus; sNd: small nematodesmata; Tc: tela corticalis.
Ultrastructure of Amylovorax
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Fig. 8. Transmission electron micrographs of A. dehorityi comb. nov. A: contractile vacuole, scale bar = 1 µm; B: contractile vacuole pore, scale bar = 500 nm. Ba: bacterium; CV: contractile vacuole; S: starch granule.
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associated fibres, the kinetodesmal fibril and the postciliary microtubular ribbon, are knitted together in alternating series to strengthen the interkinetal ridge to the right of the kinetosome. The anterior vestibulum is conical and lined down one side with vestibular cilia which enter the vestibulum as oralised invaginations of some of the anterior somatic kinetids. The opposite side of the vestibulum is lined with microtubular ribbons whose edges are connected to the vestibular wall. Cell division by telokinetal binary fission. Differential diagnosis: The amylovoracids can be distinguished from the trichostomatian order Entodiniomorphida on the basis of holotrichous somatic ciliation and simplified adoral/vestibular ciliation. The family is most similar to the holotrichous ciliate families (Isotrichidae, Paraisotrichidae and Balantiidae) currently assigned to the Order Vestibulifera. The amylovoracids differ from the balantiids by lacking a cystic transmission stage. They differ from the other exclusively endosymbiotic families on the basis of host occurrence (marsupials rather than ruminants), their ectoplasmic composition containing numerous small hydrogenosomes and apical electron dense bodies (both the isotrichids and the paraisotrichids have simple granular ectoplasms lacking these features) and the absence of large nematodesmata (found in all other trichostome families). Type genus: Amylovorax gen. nov. Other genera: Bitricha Cameron et al., 2000 is hereby removed from the Isotrichidae and redesignated as a member of the Amylovoracidae. Whilst the ultrastructural features described above which distinguish the amylovoracids from other trichostome families have yet to be demonstrated in Bitricha our phylogenetic analysis utilising small subunit rRNA gene sequences strongly supported a sister-group relationship between Amylovorax and Bitricha (Cameron et al. 2001b). The alternate hypothesis that Bitricha was part of the Isotrichidae, as proposed in Cameron et al. (2000b), received no support. For this reason we propose to redesignate Bitricha as a member of the Amylovoracidae. Habitat: Macropodid marsupial forestomach Etymology: The Amylovoracidae are named for the type genus Amylovorax. Amylovorax gen. nov. Diagnosis: With characteristics of the family. Holotrichous ciliates; somatic ciliature arranged in multiple, meridional kineties which loosely spiral
from the anterior vestibulum to the posterior of cell, in profile appearing as transverse rows descending right to left (20–30° to long axis of cell). Kineties separated evenly by uniformly spaced, regularly sized interkinetal ridges. Vestibulum small, conical and anterior. No differentiated adoral cilia, vestibular cilia short and derived from anterior end of some of the somatic kinetids. Prominent cytopharyngeal rods in most species. Single posterior contractile vacuole in most species. No obvious cytoproct. Division by equal binary fission; stomatogenesis telokinetal. Type species: Amylovorax dehorityi comb. nov. Etymology: The genus Amylovorax is named from the latin amylos (starch) and vorax (to devour) from its presumed diet of plant starch. Dasytricha dehorityi Cameron et al., 2000 is hereby redesignated as Amylovorax dehorityi comb. nov. Dasytricha dogieli Cameron et al., 2000 is hereby redesignated as Amylovorax dogieli comb. nov. Dasytricha mundayi Cameron et al., 2000 is hereby redesignated as Amylovorax mundayi comb. nov. Note: The key to species of Dasytricha included in our earlier paper (Cameron et al., 2000b) is best ignored due to the removal of half of the species considered in it to the genus Amylovorax. Amylovorax quokka sp. nov. Type host: The quokka, Setonix brachyurus (Quoy and Gaimard, 1830) Habitat: Forestomach Type locality: Rottnest Is., WA 31° 59′S 115°32′ E Prevalence: Specimens recovered from 3 (60%) of 5 of hosts examined. Type material: Holotype deposited with the Queensland Museum (Brisbane, Australia), accession number: G463131. Description (Figs. 9, 10): Body ovoid, sometimes pyriform narrowing posteriorly; 31.2–88.8 (mean 59.5) µm long by 15.2–49.6 (mean 30.0) µm wide; shape index (L/W) 1.4–2.6 (mean 2.0). Single macronucleus rod-like to spherical, most ovoid, 6.4–21.6 (mean 15.7) µm long by 4.0–11.2 (mean 7.8) µm wide, of variable location within cell. Single micronucleus, ovoid 1.6–3.2 (mean 2.3) µm long by 0.8–2.4 (mean 1.7) µm wide. Oral aperture subapical, round to oval, 3.2–5.6 (mean 3.8) µm wide . Vestibulum cup-like, 3.2–8.0 (mean 4.6) µm deep. Cytopharyngeal rods slim, 0.8–16.8 (mean 5.7) µm long, several emanate from vestibulum
Ultrastructure of Amylovorax
Fig. 9. Morphology and infraciliature of Amylovorax quokka sp. nov. A: kinety pattern; B: internal morphology. Scale bar = 10 µm. cr: cytopharyngeal rods; k: somatic kineties; ma: macronucleus; mi: micronucleus; v: vestibulum.
posteriorly. Somatic kineties with characteristics of genus, in profile 17–41 (mean 26.4) transverse (spiral) rows apparent per side, cilia 3.2–7.2 (mean 4.9) µm long. (Number of observations was 30 for all except micronucleus where n = 15.) Differential diagnosis: A. quokka sp. nov. is distinguished from A. dehorityi comb. nov. on the basis of shape, the latter species has a spindle shape and prominent mid-body kink whereas the former is ovoid to ellipsoid. A. quokka sp. nov. is distinguished from A. dogieli comb. nov. on the basis of size (A. dogieli is much larger than A. quokka) and oral aperture (in A. dogieli the aperture is set in an anterior depression whereas in A. quokka there is no depression). A. quokka sp. nov. and A. mundayi comb. nov. overlap in both size and shape but can be distinguished on the basis of wide geographic separation (offshore western Australia vs Tasmania) and differing vestibular morphology (cup-like vs conical). Etymology: A. quokka sp. nov. is named for its host the quokka, S. brachyurus.
Discussion The morphological simplicity of the group led to their misdiagnosis as isotrichids in our earlier
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paper (Cameron et al. 2000b). Indeed, no characters revealed by light microscopy distinguish them from the Isotrichidae. Even ultrastructurally, the cortical structure and kinetosome arrangement of Amylovorax dehorityi is remarkably similar to that of Dasytricha ruminantium (cf. Paul et al. 1989). Amylovorax spp. and Dasytricha spp. are all characterised by a complete body covering of dense somatic ciliation arranged in transverse (spiral) kinety rows, simple conical vestibula and no obvious cytoproct. Species in the other amylovoracid genus Bitricha resemble slightly derived isotrichids differing in their modified patterns of somatic ciliation and their ventral oral aperture. The orientation of the cells is a key difference between the genera which is not readily apparent in silverstained specimens. The vestibulum of Dasytricha is posterior and thus the oral kineties are reversed relative to trichostomes with anterior vestibula (e.g. Isotricha and Amylovorax). Overly conservative systematics has been found to be common in groups with a paucity of morphological characters (Klautau et al. 1999) and is the pitfall to which our earlier study (Cameron et al. 2000b) succumbed. The key differences, possession of ectoplasmic hydrogenosomes and simplified vestibular structure in the amylovoracids, however are significant morphological markers of the considerable evolutionary distance between the isotrichids and amylovoracids noted in our analysis of molecular sequence data (Cameron et al. 2001b). The apparent similarities between amylovoracids and isotrichids may be due to shared retention of primitive characters (symplesiomorphies) or the result of convergent evolution of derived characters (homoplasy) in two groups of stomachinhabiting ciliates. The Trichostomatia appear to be derived from a haptorian lineage by secondary loss of many cellular components including extrusomes, oral dikinetids and oral support structures. Removal of these characters from an archtypal haptorian creates a ciliate resembling a vestibuliferan, an archistome or an amylovoracid. It is probable that the simple characters shared by these groups are symplesiomorphies rather than homoplasies. While similarity of habitat could explain parallelisms between the stomach-inhabiting amylovoracids and isotrichids, it does not extend to the intestinal vestibuliferans or archistomes. Of the primitive trichostome groups, only the isotrichids are sufficiently well characterised ultrastructurally to allow valid comparisons to the
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Fig. 10. Light and electron micrographs of A. quokka sp. nov. A: mature cell, scale bar = 10 µm; B: dividing form, scale bar = 10 µm; C: scanning electron micrograph, whole cell, scale bar = 10 µm; D: scanning electron micrograph, oral zone, scale bar = 1 µm.
amylovoracids (Grain 1966; Paul et al. 1989). The kinetid structure of Dasytricha bears resemblance to that of Amylovorax in that the kinetodesmal fibril passes into the interkinetal row to its immediate right and under the postciliary ribbon of the kinetosome immediately anteriad. This produces a ‘knitted together’ appearance as the two fibres alternate down the length of the kinety linking each kinetosome with its neighbour anteriad and poste-
riad. The description of the ultrastructure of Dasytricha ruminantium by Paul et al. (1989) shows a similar alternation of the kinetodesmal fibril and postciliary microtubular ribbon in a diagram of cortical structure but no electron micrograph confirmed this arrangement. The structure of the other fibrils associated with the kinetosome was remarkably similar in the two groups, except for minor differences in the number of micro-
Ultrastructure of Amylovorax
tubules making up each fibril. The tela corticalis (frequently referred to as the ecto-endoplasmic boundary in older trichostome literature) has a similar bilayered herringbone structure in the two groups. The ectoplasms, however, are radically different. Isotrichids have hydrogenosomes confined to the endoplasm while the amylovoracids have both endo- and ectoplamsic hydrogenosomes. The apical dark bodies which line the interkinetal ridges of Amylovorax are similarly placed as the granular organelles which line the apex of the isotrichid interkinetal ridge. The two bodies, however, differ markedly in their staining characteristics (electron dense versus granular) and shape (spherical to oblong versus oval and irregular). It has been suggested that the granular organelles are secretory in nature but none are pictured discharging (Stern et al. 1977; Paul et al. 1989) and similarly none were observed discharging in this present study. They are clearly not extrusomes and a secretory function seems unlikely for the amylovoracid dark bodies due to their regular shape and nongranular structure. The degree of homology between the dark bodies and granular organelles is impossible to determine at this stage. The most significant feature of the amylovoracids is the possession of ectoplasmic hydrogenosomes. In the vast majority of ciliates, the energy producing organelles (mitochondria or hydrogenosomes) form a dense layer immediately beneath the tela corticalis or somatic cilia and are believed to provide the energy requirements of the cilia (Lynn and Corliss 1991). Neither mitochondria nor hydrogenosomes have previously been reported as occurring within the cortex of any ciliate. The position of the hydrogenosomes may be related to their function. Ectoplasmic hydrogenosomes could conceivably be more efficient in serving the energy needs of the amylovoracids than a restricted endoplasmic location. Quantitative measurements of both ciliary motion and ciliate movements are needed to evaluate whether Amylovorax species are actually more active than other ciliates. The detection of different morphotypes of hydrogenosomes in different cell compartments is unusual but these organelles have been found to be highly variable in structure in many anaerobic ciliates and flagellates (Müller 1998). Vestibular structure is another character in which ultrastructural homologies are difficult to infer between the amylovoracids and isotrichids. The basic structure of the vestibular walls in the two groups is remarkably similar with a marked separation into a
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ciliated half and a glabrous half, the latter lined with microtubular ribbons. Paul et al. (1989) suggested that the microtubule-lined portion of the vestibulum functioned as a phagoplasm and the microtubular ribbons function in the movement of food vacuoles. The microtubule-lined portions of the amylovoracid vestibulum, however, do not appear to contain significant numbers of food vacuoles. The amylovoracids similarly lacked a differentiated region of cellular ingestion; it is possible that the entire glabrous surface of the vestibulum is functioning in food uptake. The microtubular ribbons are frequently referred to in the literature (Grain 1996; Paul et al. 1989) as being derived from the transverse microtubules of the vestibular kinetosomes. Examination of amylovoracids and the published transmission electron micrographs of Grain (1966) and Paul et al. (1989) fails to show the origin of these ribbons. The ribbons are very similar to oral fibres derived from the transverse microtubular ribbon found in haptorians and they may well be homologous but their origin remains uncertain. In this study, the term “transverse microtubule” has been used so as to conform to the terminology of previous workers. Supporting the vestibulum in all trichostomatian groups is a network of fibres and nematodesmata which strengthen the structure and aid the cytoplasmic streaming necessary for ingestion. In this regard, the trend towards simplification of oral support structures seen throughout the trichostomes is most pronounced in the amylovoracids. The basic haptorian pattern comprised (in order outermost to central) nematodesmata, bulge microtubules and transverse microtubular ribbons. The trichostomes have lost the bulge microtubules and typically have highly developed basket-like arrangements of large nematodesmata. The amylovoracids lack large nematodesmata altogether. The longitudinal support fibres derived from the kinetosome bases of the vestibular and anterior somatic ciliation found in the amylovoracids are similar to the structures found in Dasytricha and identified by Paul et al. (1989) as small nematodesmata. The small nematodesamata are derived from the bases of the oral kinetosomes and apparently are modifications of the T2 microtubule. These structures are clearly different from the normal nematodesmata found in other trichostomes and haptorians which are ≥4 times the size and derived from electron-dense plates near the kinetosomes rather than from the kinetosomes themselves. The small nematodesmata have not been observed in other trichostome groups; the Entodin-
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iomorphina lack them and the Blepharocorythina and Archistomatina have yet to be examined comprehensively. An extension of the tela corticalis surrounds the vestibulum, at least apically, in the vestibuliferans and entodinio-morphs (Grain 1966; Furness and Butler 1983, 1985a, b; Paul et al. 1989; Wolska 1978a, b, c, 1979). This is not the case in the amylovoracids as the vestibulum is inserted through a hole in the tela corticalis which ends abruptly at the level of the last somatic kinetosome. The correlation between oral structures and feeding habit in ciliates has been well established (Corliss 1979). The simple diet of Dasytricha was used to explain its comparatively simple vestibular structure by Paul et al. (1989). Similarly, Furness and Butler (1988) related the relatively simple vestibulum of Entodinium, compared to other entodiniomorphs, to its limited diet. By inference, the simplicity of the amylovoracid vestibulum can be related to its simple diet which appears to be almost entirely composed of sugars and starches. It cannot be directly related to host diet; amylovoracid species have been recovered from both grazing and browsing hosts, at all times of the year despite seasonal variation in the composition of host diet, and in most geographic locations despite massive variations in location vegetation types. Any dietary specialisation by the amylovoracids reflects specialisation to particular components of the host’s diet not the specific dietary specialisations of the host itself.
Acknowledgements: The authors would like to thank Ms Kathryn Hall for drawing the diagram of the cortical fibres. This study was supported by the Australian Research Council small grants scheme and the Systematics Association grants scheme. Cameron was supported by a post-graduate scholarship from the Australian Biological Resources Study.
References Cameron S.L., O’Donoghue P.J. and Adlard R.D. (2000a): First record of Cycloposthium edentatum Strelkow, 1928 from the black-striped wallaby, Macropus dorsalis. Parasitol. Res. 86, 158–162. Cameron S.L. O’Donoghue P.J. and Adlard R.D. (2000b): Novel isotrichid ciliates endosymbiotic in Australian macropodid marsupials. Syst. Parasitol. 46, 45–57. Cameron S.L., O’Donoghue P.J. and Adlard R.D. (2001a): Four new species of Macropodinium (Ciliophora: Litostomatea) from Australian wallabies and pademelons. J. Euk. Microbiol. 48, 542–555.
Cameron S.L., Adlard R.D. and O’Donoghue P.J. (2001b): Evidence for an independent radiation of endosymbiotic litostome ciliates within Australian marsupial herbivores. Mol. Phylo. Evol. 20, 302–310. Corliss J.O. (1979): The Ciliated Protozoa, Characterisation, Classification and Guide to the Literature 2nd ed. Pergamon Press, Oxford. Dehority B.A. (1996): A new family of entodiniomorph protozoa from the marsupial forestomach, with descriptions of a new genus and five new species. J. Euk. Microbiol. 43, 285–95. Furness D.N. and Butler R.D. (1983): The cytology of sheep rumen ciliates I. Ultrastructure of Epidinium caudatum Crawley. J. Protozool. 30, 676–687. Furness D.N. and Butler R.D. (1985a): The cytology of sheep rumen ciliates II. Ultrastructure of Eudiplodinium maggii. J. Protozool. 32, 205–214. Furness D.N. and Butler R.D. (1985b): The cytology of sheep rumen ciliates III. Ultrastructure of the Genus Entodinium (Stein). J. Protozool. 32, 699–707. Furness D.N. and Butler R.D. (1988): The functional and evolutionary significance of the ultrastructure of the Ophryoscolecidae (Order Entodiniomorphida). J. Protozool. 35, 34–38. Grain J. (1966): Etude cytologique de quelques ciliés holotriches endocommensaux des ruminants et des équidés. Protistologica 2(1), 59–141; 2(2), 5–51. Ito A. and Imai S. (1998): Infraciliary bands in the rumen ophryoscolecid ciliate Ostracodinium gracile (Dogiel, 1925) observed by light microscopy. J. Euk. Microbiol. 45, 628–636. Klautau M., Russo C.A.M., Lazoski C., Boury-Esnault N., Thorpe J.P. and Solé-Cava A.M. (1999): Does cosmopolitanism result from overconservative systematics? A case study using the marine sponge Chondrilla nucula. Evolution 53, 1414–1422. Lynn D. and Corliss J. (1991): Ciliophora. In: Harrison F. and Corliss J. (eds): Microscopic Anatomy of Invertebrates. Vol. 1: Protozoa, pp. 333–467. Wiley-Liss, New York. Müller M. 1998. Enzymes and compartmentation of core energy metabolism of anaerobic protists – a special case of eukaryotic evolution? In: Coombs G.H., Vickerman K., Sleigh M.A. and Warren A. (eds): Evolutionary Relationships Among Protozoa, pp. 109–132. Chapman & Hall, London. Paul R.G., Butler R.D. and Williams A.G. (1989): Ultrastructure of the rumen ciliate Dasytricha rumantium. Europ. J. Protistol. 24, 205–215. Stern M.D., Hoover, W.H. and Leonard J.B. (1977): Ultrastructure of rumen holotrichs by electron microscopy. J. Dairy Sci. 60, 911–918. Van Hoven W., Gilchrist F. and Hamilton-Attwell V. (1987): Intestinal ciliated protozoa of African rhinoceros: Two new genera and five new species from the white rhino (Ceratotherium simum Burchell, 1817). J. Protozool. 34, 338–342. Wicklow B. J. and Hill B. F. (1992): A short procedure for protargol staining. In: Lee J. and Soldo A. (eds): Protocols in Protozoology, pp. C5.1–C5.4. Society of Protozoologists, Lawrence, KA. Wolska M. (1978a): Tripalmaria dogieli Gass., 1928 (Ciliata, Entodiniomorphida). Structure and ultrastructure Part II. Electron-microscope examinations. Acta Protozool. 17, 21–30. Wolska M. (1978b): Triadinium caudatum Fiorent. Electron microscope examinations. Acta Protozool. 17, 445–454. Wolska M. (1978c): Light and electron microscope studies on Ochoterenaia appendiculata Chavarria (Ciliata, Blepharocorythina). Acta Protozool 17, 483–492. Wolska M. (1979): Circodinium minimum (Gassovsky, 1918), Electron-microscope investigations. Acta Protozool. 18, 223–229.
The ultrastructure of Amylovorax dehorityi comb. nov. and erection of the Amylovoracidae fam. nov. (Ciliophora: Trichostomatia) Stephen L. Cameron* and Peter J. O’Donoghue Department of Microbiology & Parasitology, The University of Queensland, Brisbane 4072, Australia; E-mail: [email protected] Received: 18 June 2001; 15 October 2001; 14 November 2001. Accepted: 22 November 2001
The ultrastructural features of the holotrichous ciliates inhabiting macropodid maruspials were investigated to resolve their morphological similarity to other trichostome ciliates with observed differences in their small subunit rRNA gene sequences. The ultrastructure of Amylovorax dehorityi nov. comb. (formerly Dasytricha dehorityi) was determined by transmission electron microscopy. The somatic kineties are composed of monokinetids whose microtubules show a typical litostome pattern. The somatic cortex is composed of ridges which separate kinety rows, granular ectoplasm and a basal layer of hydrogenosomes lining the tela corticalis. The vestibulum is an invagination of the pellicle lined down one side with kineties (invaginated extensions of the somatic kineties); transverse tubules line the surface of the vestibulum and small nematodesmata surround it forming a cone-like network of struts. Cytoplasmic organelles include hydrogenosomes, irregularly shaped contractile vacuoles surrounded by a sparse spongioplasm, food vacuoles containing bacteria and large numbers of starch granules. This set of characteristics differs sufficiently from those of isotrichids and members of the genus Dasytricha to justify the erection of a new genus (Amylovorax) and a new family (Amylovoracidae). Dasytricha dehorityi, D. dogieli and D. mundayi are reassigned to the new genus Amylovorax and a new species A. quokka is erected. While the gross morphological similarities between Amylovorax and Dasytricha may be explained by convergent evolution, ultrastructural features indicate that these two genera have probably diverged independently from haptorian ancestors by successive reduction of the cortical and vestibular support structures. Key words: Amylovorax; Dasytricha; Haptoria; hydrogenosomes; Trichostomatia.
Introduction The endosymbiotic association of herbivorous mammals and trichostome ciliates is well established (Corliss 1979; Van Hoven et al. 1987; Dehority 1996). At present, 15 families of ciliates have been described within the litostome subclass Trichostomatia; all are endosymbiotic within herbivorous mammals including artiodactyls, perissodactyls, rodents, anthropoid apes and macropo*corresponding author
did marsupials. Previous investigations of the macropodid marsupials (kangaroos and wallabies) for the presence of endosymbiotic ciliates have revealed a diverse fauna including the macropodiniids (Dehority 1996; Cameron et al. 2001a), cycloposthiids (Cameron et al. 2000a) and holotrichous species originally described as isotrichids (Cameron et al. 2000b). The isotrichids had previously been considered to be confined to eutherian herbivores (ruminants and rodents) so their presence in 0932-4739/02/38/01-29 $ 15.00/0
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macropodid marsupials was surprising. Analysis of small subunit rRNA gene sequences from these Australian holotrichs suggested that their placement within the Isotrichidae was incorrect (Cameron et al. 2001b). Not only did the Australian species fail to group with the isotrichids from ruminants, they also appeared to form an independent lineage of trichostome ciliates entirely separate from those species associated with eutherian mammals. The molecular data suggested that the classification of the Australian holotrichs was wrong at the level of genus (several Australian species assigned to the ruminant-associated genus Dasytricha did not group with this genus), family (all Australian species failed to group with the isotrichids) and perhaps even order. Morphologically, however, the Australian holotrichs were almost indistinguishable from the isotrichids inhabiting ruminant when silver stained specimens were examined by light microscopy. The present study was therefore undertaken to investigate the ultrastructural features of a species of Australian holotrich (formerly classified as a member of the genus Dasytricha) and reconcile the disparate conclusions drawn from previous studies of gross morphology (Cameron et al. 2000b) and molecular sequences (Cameron et al. 2001b).
Materials and methods Samples were collected from 45 eastern-grey kangaroos (Macropus giganteus), 28 western-grey kangaroos (M. fuliginosus), 32 red-necked wallabies (M. rufogriseus) and 5 quokkas (Setonix brachyurus). Stomach fluid was removed either from road-killed animals or from animals culled from wild populations. Stomach liquor was squeezed from large fibrous matter and strained through a triple layer of surgical gauze to remove fine particulate matter and nematodes. The filtrate was fixed in 2 volumes of Bouin’s fluid for light microscopy or an equal volume of glutaraldehyde in 0.2 M cacodylate buffer for electron microscopy. Samples for transmission electron microscopy were washed 3 times with 0.2 M cacodylate buffer, 3 times in distilled water and stored in 70% ethanol. Ciliates were rehydrated, purified by Percoll density gradient centrifugation (column composed of equal volume layers of sample, 25%, 50%, 75% & 100%; spun at 3000 g for 30 min.) and individual ciliates harvested by micropipette. Cells were post-fixed in 4% osmium tetroxide (unbuffered), washed 3 times in Sorensen’s phosphate buffer (pH 7.2) and twice in distilled water. Samples were dehydrated in a graded series of acetone solutions and embedded in Epon resin; ciliates were pelleted within uncured resin by mild centrifugation. Semi-thin survey sections were cut with glass knives, stained with 1% toluidine blue and used to orientate sections. Ultrathin sections (70 nm and 90 nm) were cut with diamond knives, mounted on uncoated copper grids, stained
Table 1. Morphometric characterisation of the amylovoracid ciliate Amylovorax quokka sp. nov. recovered from the quokka, Setonix brachyurus. x: mean; SD: standard deviation; CV: coefficient of variation; min: minimum; max: maximum; n: number of observations. Character Body dimensions Length, L Width, W Shape index (L/W ratio) Macronucleus Length Width Micronucleus Length Width Oral apparatus Width Depth Length of cytopharyngeal rods Somatic ciliature Number of kineties (per side) Length of cilia
x
SD
CV
min
max
n
59.5 23.0 2.0
12.14 8.39 0.28
21.8 28.0 13.6
31.2 15.2 1.35
88.8 49.6 2.6
30 30 30
15.7 7.8
3.98 2.06
25.4 26.6
6.4 4.0
21.6 11.2
30 30
2.3 1.7
0.51 0.41
22.3 24.2
1.6 0.8
3.2 2.4
15 15
3.8 4.6 5.7
0.69 1.14 4.40
18.0 24.6 77.1
3.2 3.2 0.8
5.6 8.0 16.8
30 30 24
26.4 4.9
5.67 1.198
21.5 24.6
17 3.2
41 7.2
30 4.9
Ultrastructure of Amylovorax
with 5% uranyl acetate in 50% methanol for 6 min, washed in distilled water for 30 sec and dried. The sections were then counter-stained with Reynold’s lead citrate for 3 min, washed in distilled water for 30 sec. and dried prior to examination in a JEOL 1010 transmission electron microscope. Samples for scanning electron microscopy were washed with distilled water and purified by Percoll density gradient centrifugation as described above. Ciliates were washed 3 times in Sorensen’s phosphate buffer (pH 7.2), post-fixed in 4% osmium tetroxide (unbuffered), washed twice in water, dehydrated in a graded series of ethanol solutions and dried in a critical point drier between Millipore filters. Dried cells were sputtercoated with gold and examined in a JOEL 6300 scanning electron microscope. Samples for light microscopy were pelleted by mild centrifugation and washed 5–20 times with distilled water to remove Bouin’s fixative prior to storage in 70% ethanol. Ciliates were stained with silver proteinate (protargol) using a modification of Wicklow and Hill’s (1992) method and silver carbonate using a modification of the technique of Ito and Imai (1998). Ciliates were measured using a calibrated eyepiece micrometer and drawn with the aid of a camera lucida. Measurements are presented as a range of values followed by the arithmetic mean. Summary statistics of morphometric observations were prepared using the Statistix® program.
Results Holotrich ciliates detected in 23 (53%) of 43 eastern-grey kangaroos, 16 (62%) of 26 westerngrey kangaroos and 1 (3%) of 32 red-necked wallabies when examined by light microscopy were consistent with Cameron et al.’s (2000b) description of Dasyticha dehorityi. Holotrich ciliates detected in 3 (60%) of 5 quokkas differed sufficiently from the species of isotrichid-like ciliates recovered from macropodid marsupials to warrant the erection of a new species.
Ultrastructure Ultrastructure was studied in Dasytricha dehorityi, specimens of which were recovered from a single eastern-grey kangaroo (Macropus giganteus) collected at Mt. Sebastopol, Queensland, Australia. Somatic infraciliature Somatic ciliation is composed of monokinetids with a typical litostome pattern of a kinetodesmal fibril (opposite triplet 7), a convergent postciliary microtubular ribbon (opposite triplet 9) (Fig. 1A)
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and two transverse ribbons, the tangential transverse ribbon composed of 5 microtubules (T1) (opposite triplet 4) (Fig. 2A) and the radial transverse ribbon composed of a single microtubule (T2) (opposite triplet 5) (Fig. 2B). The kineties are arranged meridionally between pellicular projections termed the interkinetal ridges. The postciliary microtubular ribbon is composed of 3 microtubules which curve up from the kinetosome to run longitudinally and posteriorly through the right adjacent interkinetal ridge (Fig. 1B). The kinetodesmal fibril is of similar dimensions to the postciliary microtubular ribbon and curves up from the kinetosome to run laterally and forwards towards the n + 1 kinetosome of the adjacent right kinety (Fig. 2A). The kinetodesmal fibril passes under the postciliary ribbon of the kinetosome immediately anteriorad and is anchored in the interkinetal ridge to the immediate right. The T1 fibre curves up and anteriorly to form the right-most edge of the interkinetal ridge to the immediate left of the kinetosome. The T2 fibre is curved down and slightly anteriorly as it pierces the tela corticalis (Fig. 2B). A diagram of the three dimensional arrangement of the kineties is presented in Figure 3. Cortex The interkinetal ridges have a prominent epiplasm composed of small flattened sacs (Fig. 4A). The ridges have an apical layer of electron-dense oblong bodies placed perpendicularly to the cell membrane (Fig. 2B). These bodies vary in size from 250–300 nm but are always present as a single layer (Fig. 4B). The postciliary ribbon passes over the top of the layer separating the oblong bodies from the cell membrane (not shown). The kinetodesmal fibril passes between adjacent bodies to anchor in the apex of the interkinetal ridge (Fig. 2A). The main body of the interkinetal ridges and the region between the kinetosomes is composed of a granular ectoplasm with few inclusions (Fig. 2A). The base of the ectoplasm is made up of small ovoid hydrogenosomes which carpet the ectoplasmic surface of the tela corticalis (Fig. 4B). The tela corticalis is composed of a double layer of microfilaments which have a herringbone appearance in cross section. There is a subcortical layer composed of small, empty, membrane-bound vacuoles separating the tela corticalis from the endoplasm (Fig. 5A).
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Fig. 1. Transmission electron micrographs of A. dehorityi comb. nov. A: transverse section through somatic kinetids, scale bar = 500 nm; B: longitudinal section through somatic kinety, scale bar = 1 µm. H1: endoplasmic hydrogenosomes; H2: ectoplasmic hydrogenosomes; Kd: kinetodesmal fibril; Pc: postciliary microtubular ribbon; S: starch granule; Tc: tela corticalis.
Ultrastructure of Amylovorax
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Fig. 2. Transmission electron micrographs of A. dehorityi comb. nov. A: glancing section through somatic kineties, scale bar = 500 nm; B: glancing section through somatic kineties, scale bar = 500 nm. Db: dark body; Kd: kinetodesmal fibril; Pc: postciliary microtubular ribbon; Ps: pallicular space; T1: tangential transverse ribbon; T2: radial transverse ribbon; Tc: tela corticalis.
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with the vestibular kinetids. The vestibulum thus forms a cone which pierces the tela corticalis. There is no differentiated phagoplasm; no obvious structure separates the vestibular associated microtubules from the endoplasmic organelles.
Fig. 3. Three dimensional arrangement of cortex fibres of A. dehorityi comb. nov. Kd: kinetodesmal fibril; Pc: postciliary microtubular ribbon; T1: tangential transverse ribbon. The arrow points towards the anterior end of the cell.
Vestibulum and oral infraciliature The vestibulum is a simple conical invagination of the apical end of the cell (Fig. 5B). It is lined along one side by several kineties which are invaginated extensions of the somatic kineties. The opposite side of the vestibulum lacks cilia and is lined with evenly spaced microtubular ribbons set perpendicularly to the vestibulum wall (Fig. 5B). The vestibular kineties are separated by interkinetal ridges which are of similar structure to the somatic interkinetal ridges except that they lack the apical layer of oblong bodies (Fig. 6A). Transverse tubules associated with the bases of the kinetosomes are similar in structure to those associated with the somatic kinetosomes and are derived from the T1 microtubule (Fig. 6B). Transverse tubules also line the glabrous dorsal side of the vestibulum (Fig. 6B). The small nematodesmata are derived from an electron-dense plate at the base of the kinetosomes of the vestibular kinetids and the anterior-most kinetosomes of the somatic kinetids. The small nematodesmata are composed of 4 to 6 microtubules which pierce the tela corticalis, enter the endoplasm (Fig. 6A) and run parallel to the vestibulum, surrounding it and forming a cone-like network of struts (Fig. 7A). The tela corticalis does not line the vestibulum internally, it stops abruptly at the level of the anterior-most kinetosome of the somatic kinetids not associated
Organelles The macronucleus is surrounded by a porous nuclear envelope and contains chromatin bodies which have condensed away from the envelope (Fig. 7B). The micronucleus has a different structure; the chromatin has condensed into a single central body with a striated appearance (Fig. 7B). The endoplasm is dominated by the starch granules which make up a majority of the cell contents (Fig. 8A). There are a few food vacuoles containing bacteria. The hydrogenosomes appear to be of two types; the ectoplasm has a basal layer composed of small ovoid hydrogenosomes while the endoplasmic hydrogenosomes are larger and more irregular in shape (Fig. 4B). There is a little endoplasmic reticulum around the periphery of the endoplasm near the tela corticalis (Fig. 4A). There is a single posterior contractile vacuole which is frequently oblong or irregular in shape. There is a sparse amount of spongioplasm which does not differ greatly from the regular endoplasm (Fig. 8A). The contractile vacuole is endoplasmic; the duct pierces the tela corticalis and empties between adjacent interkinetal ridges. The duct is lined with microtubules for rigidity (Fig. 8B) and there are no accessory collecting tubules.
Taxonomy The ultrastructural features of these ciliates were sufficiently novel (cortex structure and vestibular arrangement) to justify the formal erection of a new family, as follows. Family Amylovoracidae fam. nov. Trichostomatia incertae sedis Diagnosis: Cells oval, elongate, pyriform or falcate. Somatic ciliation holotrichous with meridional kineties arranged between pellicular interkinetal ridges. Most species have prominent cytopharyngeal rods. Single macronucleus; single micronucelus generally adjacent to the macronucleus. A prominent tela corticalis divides the cell into endoplasm and ectoplasm. The ectoplasm is lined basally by small hydrogenosomes and apically by small electron-dense bodies. The kinetosome-
Ultrastructure of Amylovorax
35
Fig. 4. Transmission electron micrographs of A. dehorityi comb. nov. A: glancing section through kineties, scale bar = 500 nm; B: longitudinal section through interkinetal ridge, scale bar = 500 nm. Db: dark body; H1: endoplasmic hydogenosomes; H2: ectoplasmic hydogenosomes; Ps: pallicular space; Tc: tela corticalis.
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Fig. 5. Transmission electron micrographs of A. dehorityi comb. nov. A: longitudinal section through kinety, scale bar = 500 nm; B: sagittal section through vestibulum, scale bar = 1 µm. ER: endoplasmic reticulum; Tc: tela corticalis; V: vacuole.
Ultrastructure of Amylovorax
37
Fig. 6. Transmission electron micrographs of A. dehorityi comb. nov. A: oralised kineties, scale bar = 200 nm; B: sagital section though dorsal vestibulum, scale bar = 500 nm. sNd: small nematodesmata; T1: tangential transverse ribbon; TT: transverse tubules; Pl: electron-dense plate.
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Fig. 7. Transmission electron micrographs of A. dehorityi comb. nov. A: glancing section through forebody, scale bar = 2 µm; B: nuclei, scale bar = 1 µm. C: chromatin ring; Ma: macronucleus; Mi: micronucleus; sNd: small nematodesmata; Tc: tela corticalis.
Ultrastructure of Amylovorax
39
Fig. 8. Transmission electron micrographs of A. dehorityi comb. nov. A: contractile vacuole, scale bar = 1 µm; B: contractile vacuole pore, scale bar = 500 nm. Ba: bacterium; CV: contractile vacuole; S: starch granule.
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associated fibres, the kinetodesmal fibril and the postciliary microtubular ribbon, are knitted together in alternating series to strengthen the interkinetal ridge to the right of the kinetosome. The anterior vestibulum is conical and lined down one side with vestibular cilia which enter the vestibulum as oralised invaginations of some of the anterior somatic kinetids. The opposite side of the vestibulum is lined with microtubular ribbons whose edges are connected to the vestibular wall. Cell division by telokinetal binary fission. Differential diagnosis: The amylovoracids can be distinguished from the trichostomatian order Entodiniomorphida on the basis of holotrichous somatic ciliation and simplified adoral/vestibular ciliation. The family is most similar to the holotrichous ciliate families (Isotrichidae, Paraisotrichidae and Balantiidae) currently assigned to the Order Vestibulifera. The amylovoracids differ from the balantiids by lacking a cystic transmission stage. They differ from the other exclusively endosymbiotic families on the basis of host occurrence (marsupials rather than ruminants), their ectoplasmic composition containing numerous small hydrogenosomes and apical electron dense bodies (both the isotrichids and the paraisotrichids have simple granular ectoplasms lacking these features) and the absence of large nematodesmata (found in all other trichostome families). Type genus: Amylovorax gen. nov. Other genera: Bitricha Cameron et al., 2000 is hereby removed from the Isotrichidae and redesignated as a member of the Amylovoracidae. Whilst the ultrastructural features described above which distinguish the amylovoracids from other trichostome families have yet to be demonstrated in Bitricha our phylogenetic analysis utilising small subunit rRNA gene sequences strongly supported a sister-group relationship between Amylovorax and Bitricha (Cameron et al. 2001b). The alternate hypothesis that Bitricha was part of the Isotrichidae, as proposed in Cameron et al. (2000b), received no support. For this reason we propose to redesignate Bitricha as a member of the Amylovoracidae. Habitat: Macropodid marsupial forestomach Etymology: The Amylovoracidae are named for the type genus Amylovorax. Amylovorax gen. nov. Diagnosis: With characteristics of the family. Holotrichous ciliates; somatic ciliature arranged in multiple, meridional kineties which loosely spiral
from the anterior vestibulum to the posterior of cell, in profile appearing as transverse rows descending right to left (20–30° to long axis of cell). Kineties separated evenly by uniformly spaced, regularly sized interkinetal ridges. Vestibulum small, conical and anterior. No differentiated adoral cilia, vestibular cilia short and derived from anterior end of some of the somatic kinetids. Prominent cytopharyngeal rods in most species. Single posterior contractile vacuole in most species. No obvious cytoproct. Division by equal binary fission; stomatogenesis telokinetal. Type species: Amylovorax dehorityi comb. nov. Etymology: The genus Amylovorax is named from the latin amylos (starch) and vorax (to devour) from its presumed diet of plant starch. Dasytricha dehorityi Cameron et al., 2000 is hereby redesignated as Amylovorax dehorityi comb. nov. Dasytricha dogieli Cameron et al., 2000 is hereby redesignated as Amylovorax dogieli comb. nov. Dasytricha mundayi Cameron et al., 2000 is hereby redesignated as Amylovorax mundayi comb. nov. Note: The key to species of Dasytricha included in our earlier paper (Cameron et al., 2000b) is best ignored due to the removal of half of the species considered in it to the genus Amylovorax. Amylovorax quokka sp. nov. Type host: The quokka, Setonix brachyurus (Quoy and Gaimard, 1830) Habitat: Forestomach Type locality: Rottnest Is., WA 31° 59′S 115°32′ E Prevalence: Specimens recovered from 3 (60%) of 5 of hosts examined. Type material: Holotype deposited with the Queensland Museum (Brisbane, Australia), accession number: G463131. Description (Figs. 9, 10): Body ovoid, sometimes pyriform narrowing posteriorly; 31.2–88.8 (mean 59.5) µm long by 15.2–49.6 (mean 30.0) µm wide; shape index (L/W) 1.4–2.6 (mean 2.0). Single macronucleus rod-like to spherical, most ovoid, 6.4–21.6 (mean 15.7) µm long by 4.0–11.2 (mean 7.8) µm wide, of variable location within cell. Single micronucleus, ovoid 1.6–3.2 (mean 2.3) µm long by 0.8–2.4 (mean 1.7) µm wide. Oral aperture subapical, round to oval, 3.2–5.6 (mean 3.8) µm wide . Vestibulum cup-like, 3.2–8.0 (mean 4.6) µm deep. Cytopharyngeal rods slim, 0.8–16.8 (mean 5.7) µm long, several emanate from vestibulum
Ultrastructure of Amylovorax
Fig. 9. Morphology and infraciliature of Amylovorax quokka sp. nov. A: kinety pattern; B: internal morphology. Scale bar = 10 µm. cr: cytopharyngeal rods; k: somatic kineties; ma: macronucleus; mi: micronucleus; v: vestibulum.
posteriorly. Somatic kineties with characteristics of genus, in profile 17–41 (mean 26.4) transverse (spiral) rows apparent per side, cilia 3.2–7.2 (mean 4.9) µm long. (Number of observations was 30 for all except micronucleus where n = 15.) Differential diagnosis: A. quokka sp. nov. is distinguished from A. dehorityi comb. nov. on the basis of shape, the latter species has a spindle shape and prominent mid-body kink whereas the former is ovoid to ellipsoid. A. quokka sp. nov. is distinguished from A. dogieli comb. nov. on the basis of size (A. dogieli is much larger than A. quokka) and oral aperture (in A. dogieli the aperture is set in an anterior depression whereas in A. quokka there is no depression). A. quokka sp. nov. and A. mundayi comb. nov. overlap in both size and shape but can be distinguished on the basis of wide geographic separation (offshore western Australia vs Tasmania) and differing vestibular morphology (cup-like vs conical). Etymology: A. quokka sp. nov. is named for its host the quokka, S. brachyurus.
Discussion The morphological simplicity of the group led to their misdiagnosis as isotrichids in our earlier
41
paper (Cameron et al. 2000b). Indeed, no characters revealed by light microscopy distinguish them from the Isotrichidae. Even ultrastructurally, the cortical structure and kinetosome arrangement of Amylovorax dehorityi is remarkably similar to that of Dasytricha ruminantium (cf. Paul et al. 1989). Amylovorax spp. and Dasytricha spp. are all characterised by a complete body covering of dense somatic ciliation arranged in transverse (spiral) kinety rows, simple conical vestibula and no obvious cytoproct. Species in the other amylovoracid genus Bitricha resemble slightly derived isotrichids differing in their modified patterns of somatic ciliation and their ventral oral aperture. The orientation of the cells is a key difference between the genera which is not readily apparent in silverstained specimens. The vestibulum of Dasytricha is posterior and thus the oral kineties are reversed relative to trichostomes with anterior vestibula (e.g. Isotricha and Amylovorax). Overly conservative systematics has been found to be common in groups with a paucity of morphological characters (Klautau et al. 1999) and is the pitfall to which our earlier study (Cameron et al. 2000b) succumbed. The key differences, possession of ectoplasmic hydrogenosomes and simplified vestibular structure in the amylovoracids, however are significant morphological markers of the considerable evolutionary distance between the isotrichids and amylovoracids noted in our analysis of molecular sequence data (Cameron et al. 2001b). The apparent similarities between amylovoracids and isotrichids may be due to shared retention of primitive characters (symplesiomorphies) or the result of convergent evolution of derived characters (homoplasy) in two groups of stomachinhabiting ciliates. The Trichostomatia appear to be derived from a haptorian lineage by secondary loss of many cellular components including extrusomes, oral dikinetids and oral support structures. Removal of these characters from an archtypal haptorian creates a ciliate resembling a vestibuliferan, an archistome or an amylovoracid. It is probable that the simple characters shared by these groups are symplesiomorphies rather than homoplasies. While similarity of habitat could explain parallelisms between the stomach-inhabiting amylovoracids and isotrichids, it does not extend to the intestinal vestibuliferans or archistomes. Of the primitive trichostome groups, only the isotrichids are sufficiently well characterised ultrastructurally to allow valid comparisons to the
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S. L. Cameron and P. J. O’Donoghue
Fig. 10. Light and electron micrographs of A. quokka sp. nov. A: mature cell, scale bar = 10 µm; B: dividing form, scale bar = 10 µm; C: scanning electron micrograph, whole cell, scale bar = 10 µm; D: scanning electron micrograph, oral zone, scale bar = 1 µm.
amylovoracids (Grain 1966; Paul et al. 1989). The kinetid structure of Dasytricha bears resemblance to that of Amylovorax in that the kinetodesmal fibril passes into the interkinetal row to its immediate right and under the postciliary ribbon of the kinetosome immediately anteriad. This produces a ‘knitted together’ appearance as the two fibres alternate down the length of the kinety linking each kinetosome with its neighbour anteriad and poste-
riad. The description of the ultrastructure of Dasytricha ruminantium by Paul et al. (1989) shows a similar alternation of the kinetodesmal fibril and postciliary microtubular ribbon in a diagram of cortical structure but no electron micrograph confirmed this arrangement. The structure of the other fibrils associated with the kinetosome was remarkably similar in the two groups, except for minor differences in the number of micro-
Ultrastructure of Amylovorax
tubules making up each fibril. The tela corticalis (frequently referred to as the ecto-endoplasmic boundary in older trichostome literature) has a similar bilayered herringbone structure in the two groups. The ectoplasms, however, are radically different. Isotrichids have hydrogenosomes confined to the endoplasm while the amylovoracids have both endo- and ectoplamsic hydrogenosomes. The apical dark bodies which line the interkinetal ridges of Amylovorax are similarly placed as the granular organelles which line the apex of the isotrichid interkinetal ridge. The two bodies, however, differ markedly in their staining characteristics (electron dense versus granular) and shape (spherical to oblong versus oval and irregular). It has been suggested that the granular organelles are secretory in nature but none are pictured discharging (Stern et al. 1977; Paul et al. 1989) and similarly none were observed discharging in this present study. They are clearly not extrusomes and a secretory function seems unlikely for the amylovoracid dark bodies due to their regular shape and nongranular structure. The degree of homology between the dark bodies and granular organelles is impossible to determine at this stage. The most significant feature of the amylovoracids is the possession of ectoplasmic hydrogenosomes. In the vast majority of ciliates, the energy producing organelles (mitochondria or hydrogenosomes) form a dense layer immediately beneath the tela corticalis or somatic cilia and are believed to provide the energy requirements of the cilia (Lynn and Corliss 1991). Neither mitochondria nor hydrogenosomes have previously been reported as occurring within the cortex of any ciliate. The position of the hydrogenosomes may be related to their function. Ectoplasmic hydrogenosomes could conceivably be more efficient in serving the energy needs of the amylovoracids than a restricted endoplasmic location. Quantitative measurements of both ciliary motion and ciliate movements are needed to evaluate whether Amylovorax species are actually more active than other ciliates. The detection of different morphotypes of hydrogenosomes in different cell compartments is unusual but these organelles have been found to be highly variable in structure in many anaerobic ciliates and flagellates (Müller 1998). Vestibular structure is another character in which ultrastructural homologies are difficult to infer between the amylovoracids and isotrichids. The basic structure of the vestibular walls in the two groups is remarkably similar with a marked separation into a
43
ciliated half and a glabrous half, the latter lined with microtubular ribbons. Paul et al. (1989) suggested that the microtubule-lined portion of the vestibulum functioned as a phagoplasm and the microtubular ribbons function in the movement of food vacuoles. The microtubule-lined portions of the amylovoracid vestibulum, however, do not appear to contain significant numbers of food vacuoles. The amylovoracids similarly lacked a differentiated region of cellular ingestion; it is possible that the entire glabrous surface of the vestibulum is functioning in food uptake. The microtubular ribbons are frequently referred to in the literature (Grain 1996; Paul et al. 1989) as being derived from the transverse microtubules of the vestibular kinetosomes. Examination of amylovoracids and the published transmission electron micrographs of Grain (1966) and Paul et al. (1989) fails to show the origin of these ribbons. The ribbons are very similar to oral fibres derived from the transverse microtubular ribbon found in haptorians and they may well be homologous but their origin remains uncertain. In this study, the term “transverse microtubule” has been used so as to conform to the terminology of previous workers. Supporting the vestibulum in all trichostomatian groups is a network of fibres and nematodesmata which strengthen the structure and aid the cytoplasmic streaming necessary for ingestion. In this regard, the trend towards simplification of oral support structures seen throughout the trichostomes is most pronounced in the amylovoracids. The basic haptorian pattern comprised (in order outermost to central) nematodesmata, bulge microtubules and transverse microtubular ribbons. The trichostomes have lost the bulge microtubules and typically have highly developed basket-like arrangements of large nematodesmata. The amylovoracids lack large nematodesmata altogether. The longitudinal support fibres derived from the kinetosome bases of the vestibular and anterior somatic ciliation found in the amylovoracids are similar to the structures found in Dasytricha and identified by Paul et al. (1989) as small nematodesmata. The small nematodesamata are derived from the bases of the oral kinetosomes and apparently are modifications of the T2 microtubule. These structures are clearly different from the normal nematodesmata found in other trichostomes and haptorians which are ≥4 times the size and derived from electron-dense plates near the kinetosomes rather than from the kinetosomes themselves. The small nematodesmata have not been observed in other trichostome groups; the Entodin-
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iomorphina lack them and the Blepharocorythina and Archistomatina have yet to be examined comprehensively. An extension of the tela corticalis surrounds the vestibulum, at least apically, in the vestibuliferans and entodinio-morphs (Grain 1966; Furness and Butler 1983, 1985a, b; Paul et al. 1989; Wolska 1978a, b, c, 1979). This is not the case in the amylovoracids as the vestibulum is inserted through a hole in the tela corticalis which ends abruptly at the level of the last somatic kinetosome. The correlation between oral structures and feeding habit in ciliates has been well established (Corliss 1979). The simple diet of Dasytricha was used to explain its comparatively simple vestibular structure by Paul et al. (1989). Similarly, Furness and Butler (1988) related the relatively simple vestibulum of Entodinium, compared to other entodiniomorphs, to its limited diet. By inference, the simplicity of the amylovoracid vestibulum can be related to its simple diet which appears to be almost entirely composed of sugars and starches. It cannot be directly related to host diet; amylovoracid species have been recovered from both grazing and browsing hosts, at all times of the year despite seasonal variation in the composition of host diet, and in most geographic locations despite massive variations in location vegetation types. Any dietary specialisation by the amylovoracids reflects specialisation to particular components of the host’s diet not the specific dietary specialisations of the host itself.
Acknowledgements: The authors would like to thank Ms Kathryn Hall for drawing the diagram of the cortical fibres. This study was supported by the Australian Research Council small grants scheme and the Systematics Association grants scheme. Cameron was supported by a post-graduate scholarship from the Australian Biological Resources Study.
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