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175 Abstract The Class ARMOPHOREA represents a new assemblage of ciliates, and one of the two “ riboclasses” as their establishment is completely dependent upon small subunit rRNA gene sequences that showed affi nities of the two included orders – Armophorida and Clevelandellida. The ciliates in this class occupy anoxic habitats. Armophorids are typical of sapropelic habitats, but can be benthic or planktonic, while clevelandellids are endosymbi- onts in the digestive systems of a wide variety of invertebrates, particularly insects, and some verte- brates, particularly amphibians. While their somatic dikinetids are quite different, armophoreans are all characterized by having their mitochondria trans- formed to hydrogenosomes, organelles that provide hydrogen to the methanogenic bacterial symbionts of these ciliates. The oral structures of the two orders are also divergent: membranelle-like in armophorids and heteromembranelles in clevelandellids. Stomato- genesis is pleurotelokinetal. The macronucleus is of simple form, but polytene chromosomes develop after conjugation and the macronuclear DNA ulti- mately differentiates into gene-sized pieces. Armo- phorids, because of their habitat preferences, are particularly good bioindicators of anoxic aquatic environments. Keywords Endosymbiont, cathetodesmal fi bril, sulfureta, secant system The ciliates included in this class are typically small to medium-sized cells. Armophoreans are free- swimming and typically holotrichously ciliated. However, their body ciliation can vary from many, densely ciliated kineties in some clevelandellids to only anterior and posterior cirrus-like tufts in some armophorids. All species have multiple adoral polykinetids, ranging from around a dozen in some armophorids to several dozens in some clevelandellids . These ciliates are very restricted in their distribution. Although world-wide, they are confined to sediments, both aquatic (Fenchel, 1993) and terrestrial (Foissner, 1987), and the water column (Fenchel et al., 1995), where oxygen tensions are extremely reduced to absent. They are also found as endocommensal symbionts in the digestive tracts of a variety of metazoans, rang- ing from selected invertebrates (Albaret, 1970b; Hackstein & Stumm, 1994) through to amphibians (Affa’a, Ndongo, & Granosik, 1995). Interest has increased in the group recently because they harbor endosymbiotic methanogenic bacteria , which can themselves produce the greenhouse gas, methane . There can be thousands of methanogenic bacteria per ciliate (van Bruggen, Stumm, & Vogels, 1983), producing significant quantities of methane , which is then liberated into the environment (Fenchel & Finlay, 1992; Hackstein & Stumm, 1994). The name of the class, ARMOPHOREA , is derived from the subordinal name originally pro- posed by Jankowski (1964a, 1964b) to include only the caenomorphid heterotrichs, which he argued derived from a Metopus -like ancestor. It derives from the Latin arma , meaning weapons (or it derives from the Latin armus meaning shoulder), and refers to the fact that caenomorphids have the appearance of military helmets (or the caenomor- phid body is twisted to give the appearance of a shoulder). Although not highly similar, a number Chapter 8 Subphylum 2. INTRAMACRONUCLEATA: Class 2. ARMOPHOREA – Sapropelibionts that Once Were Heterotrichs 176 8. Subphylum 2. INTRAMACRONUCLEATA: Class 2. ARMOPHOREA of clevelandellids have conspicuous polysaccha- ride “skeletal” elements in their cortex, an “armor” of a different sort (see Albaret, 1970a). Like the Class SPIROTRICHEA , there is no conspicuous synapomorphy for members of this class. They are united by the following three fea- tures. First, they are restricted to anaerobic habitats and are typically dependent upon methanogenic symbionts . Although this is not a unique feature for the Class ARMOPHOREA (see particularly Chapter 12. Class PLAGIOPYLEA ), we predict that the metabolic dependence on hydrogenases in this class will be shown to have a common phylogenetic origin. Second, clevelandellids and armophorids share pleurotelokinetal stomatogen- esis of the adoral polykinetids , a feature shown by members of other classes (Foissner & Agatha, 1999). Finally, they show strong similarities in the sequences of their small subunit rRNA (SSUrRNA) genes (Embley et al., 1995; Hackstein, Van Hoek, Leunissen, & Huynen, 2001; van Hoek, van Alen, Sprakel, Hackstein, & Vogels, 1998). This class could be called the first “ riboclass ” of ciliates, since its monophyly is predicted by sequence analyses of the SSUrRNA genes. However, we do not yet have a signature sequence that would char- acterize the class. 8.1 Taxonomic Structure The two major groups – the clevelandellids and armophorids – included in this class have long been considered heterotrichs because of their pos- session of multiple adoral polykinetids (Fig. 8.1). Corliss (1979) considered them to be suborders within the Order Heterotrichida . However, early ultrastructural analysis demonstrated clear differ- ences between the somatic and oral structures of clevelandellids and their presumed “ heterotrich ” relatives. The somatic dikinetids do not give rise to postciliodesmata , their kinetodesmal fibril is differ- ently shaped, and there is a prominent left-directed striated cathetodesmal fibril arising adjacent to the anterior kinetosome (Paulin, 1967; de Puytorac & Grain, 1969, 1976). Although there is still no pub- lished account devoted solely to the ultrastructure of armophorids , Schrenk and Bardele (1991) have indicated differences between the somatic kinetid of the armophorid Metopus and those of cleve- landellids . It does appear that Metopus may have cathetodesmal-like fibrils , which do not appear striated. Little research has been done on members of this class, outside the recent interest in their symbiotic methanogens (see below Life History and Ecology ). We place armophorids and clevelandellids in the Class ARMOPHOREA primarily based on their strong association derived from sequence simi- larities of the SSUrRNA gene: the clevelandellids Nyctotherus and Nyctotheroides strongly group with the armophorids Metopus and Caenomorpha (Embley et al., 1995; van Hoek et al., 1998). Both Jankowski (1968b) and Albaret (1975) have sug- gested that clevelandellids may have derived from metopids through transformation of the cortical patterning, following a suggestion by Villeneuve- Brachon (1940). Therefore, we place these two groups together and elevate them to ordinal status, as others have done (Lynn & Small, 1997, 2002; de Puytorac, 1994a; Small & Lynn, 1985). Following Jankowski (1964a, 1964b, 1968b) and Albaret (1975), we assume that the free-living armophorids represent the descendants of the ancestral group from which the endosymbiotic cleve- landellids evolved. The Order Armophorida includes two fami- lies: the Family Metopidae and the Family Caenomorphidae (Fig. 8.1). In most forms, there is a slight twist left to the anterior end of the body, which is covered by up to five perizonal or epistomial kineties (e.g., Fernández-Galiano & Fernández-Leborans, 1980; Jankowski, 1968b). This twist becomes pronounced in derived forms and in all caenomorphids (Fig. 8.1). Caenomorphids are not typically holotrichous, but rather may have the somatic ciliation restricted to anterior and pos- terior cirrus-like tufts. The Order Clevelandellida has not changed in composition since Corliss (1979). It contains five families: the Family Nyctotheridae , the Family Sicuophoridae , the Family Clevelandellidae , the Family Inferostomatidae , and the Family Nathellidae . The latter two families are mono- typic. Clevelandellids are densely ciliated, often laterally compressed ciliates with manyleft serial oral polykinetids that are hidden in a groove-like peristome and deep oral cavity or infundibulum (Fig. 8.1). These obligate endosymbionts are com- mensal in a wide range of hosts: Nyctotherus is Fig. 8.1. Stylized drawings of representative genera from the two orders in the Class ARMOPHOREA . Order Armophorida : the metopids Bothrostoma and Metopus , and the caenomorphid Caenomorpha . Order Clevelandellida : Nyctotherus and Clevelandella 8.1 Taxonomic Structure 177 178 8. Subphylum 2. INTRAMACRONUCLEATA: Class 2. ARMOPHOREA found in oligochaetes , insects , and myriapods ; Nyctotheroides is found in frogs and toads ; and Clevelandella is found in wood roaches and ter- mites (see Life History and Ecology ). Systematic research on members of this group has been done by literally a handful of investigators, following monographic work on the armophorids and caenomorphids by Jankowski (1964a, 1964b) and on the clevelandellids by Albaret and coworkers (Albaret, 1975; Albaret & Njiné, 1976). Exploration of the biodiversity of clevelandellid symbionts of African anurans has been expanded considerably by Affa’a (1980, 1983, 1988b) while Affa’a (1989) and Grim (1992) have described new genera sym- biotic in fishes (see also Earl, 1991). Esteban, Fenchel, and Finlay (1995) have taken a conservative approach in their revision of Metopus , reducing 76 nominal species to 22 mor- phospecies. It will be up to molecular systematists to determine if these morphospecies are really as phenotypically variable as presumed by Esteban et al. (1995). 8.2 Life History and Ecology Armophoreans, like most ciliates, are globally dis- tributed. A novel technical approach to their study used electromigration to extract these often sedi- ment-dwelling ciliates from their habitats (Wagener, Stumm, & Vogels, 1986). Free-living armo phorids have been found in freshwater and marine habitats in Eurasia (e.g., Agamaliev, 1974; Finlay & Maberly, 2000; Grolière & Njiné, 1973; Guhl, Finlay, & Schink, 1996; Madoni & Sartore, 2003) and North America (Bamforth, 1963; Borror, 1963), and chloride lakes (Madoni, 1990). In these habitats, they are part of the sulfureta community, which may also include ciliates from the Classes HETEROTRICHEA , PLAGIOPYLEA , and OLIGO HYMENOPHOREA (Dyer, 1989; Fenchel, 1987). Foissner (1987, 1995b) recorded metopids from temperate and tropical soils in which they survive by encystment. Encystment is crucial to the transmission between hosts of the clevelandellids, all of which are endosymbionts in both terrestrial and aquatic metazoans. These ciliates have been recorded from diverse hosts: insects (Hackstein & Stumm, 1994; Lalpotu, 1980a, 1980b; Zeliff, 1933), millipedes (Albaret, 1970b; Hackstein & Stumm; Lalpotu, 1980c), molluscs (Laval & Tuffrau, 1973), sea urchins (Biggar & Wenrich, 1932; Grolière, de Puytorac, & Grain, 1980b), fishes (Grim, 1998; Grim, Clements, & Byfield, 2002; Grim, Reed, & Fishelson, 1995/1996; Jankowski, 1974a), amphibians (Albaret, 1975; Affa’a et al., 1995; Wilbert & Schmeier, 1982), and reptiles (Geiman & Wichterman, 1937; Takahashi & Imai, 1989). Free-living armophorids are restricted to anoxic or microaerobic habitats, such as the anoxic hypo- limnion in lakes and bays or the anoxic layers in sediments. The armophorids Caenomorpha and Metopus can reach abundances of more than 5,000 l −1 in the water column, but are typically much less abundant than this (Fenchel & Finlay, 1991a; Fenchel, Kristensen, & Rasmussen, 1990; Guhl & Finlay, 1993; Guhl et al., 1996). Armophorids increase their relative abundance in sediments during periods of anoxia, reaching more than 50 ml −1 of sediment (Fenchel, 1993; Finlay, 1982). These ciliates survive best at low oxygen concentrations. They exhibit a chemosensory response to oxygen concentration: they increase their swimming speed at higher oxygen concentrations and show ciliary reversals when leaving anoxic conditions and enter- ing an oxygen zone (Fenchel & Finlay, 1990a). The abundances of symbiotic clevelandellids depend partly on the host. Wilbert and Schmeier (1982) recorded hundreds of Nyctotheroides in some frog hosts while Gijzen and Barugahare (1992) recorded over 10 4 ml −1 Nyctotherus in the hindgut of the American cockroach Periplaneta americana . Armophoreans typically feed on heterotrophic and phototrophic purple bacteria , and typically grow more slowly than comparably-sized aerobic ciliates with generation times in the order of days (Fenchel & Finlay, 1990b). Metopus requires bacte- rial abundances of more than 10 7 ml −1 for maximum growth (Massana, Stumm, & Pedrós-Alió, 1994). The abundance of Caenomorpha is correlated with the abundance of its photosynthetic bacterial prey, Thiopedia , suggesting that there is chemosensory tracking of prey by this ciliate predator (Guhl & Finlay, 1993). While Guhl and Finlay (1993) con- cluded that Thiopedia production is controlled by Caenomorpha , Massana and Pedrós-Alió (1994) concluded in another habitat that anaerobic cili- ates do not likely control bacterial production. The growth efficiencies of anaerobic ciliates are quite low, less than 10%. Although these ciliates are not dependent upon their intracellular endosymbiotic methanogenic bacteria, their growth rates can, in some cases, be reduced if deprived of their bacteria. Although there is yet no direct evidence, the metha- nogens in these cases may be supplying the host ciliate with organic excretions to enhance the growth rate (Fenchel & Finlay, 1991b). One of the first surveys of symbiotic bacteria was that of Fenchel, Perry, and Thane (1977) who reported both ectosymbiotic and endosymbiotic bacteria in the armophoreans Caenomorpha and Metopus . Endosymbiotic methanogenic bacteria have been reported in members of both orders of armophoreans (e.g., Fenchel & Finlay, 1991a; Gijzen & Barugahare, 1992). Many of these bacteria have been confirmed to be methanogens , which can number from hundreds to over 8,000 per ciliate (Fenchel, 1993). They can take various shapes from elongate rods, up to 7 µm in length, to coccoid forms, about 0.5 µm in diameter. Methanogens were identified first on the basis of a characteristic, fluo- rescent, deazaflavin coenzyme F 420 (van Bruggen et al., 1983). Van Bruggen, Zwart, van Assema, Stumm, and Vogels (1984) and Van Bruggen et al. (1986) were first to isolate and characterize the methanogens to the genera Methanobacterium and Methanoplanus . Use of the polymerase chain reaction has increased the diversity of methano- gens to include potentially other genera, such as Methanolobus and Methanocorpusculum (Embley & Finlay, 1994). In both free-living and symbiotic armophoreans , unrelated ciliates may contain the same methanogen species while the same ciliate species may at different times or in different hosts carry different methanogen species. This demon- strates that losses and acquisitions of methanogens are continually occurring and some may be quite recent acquisitions (Embley & Finlay, 1993; van Hoek et al., 2000b). We do not yet know how the association is established since the bacteria lie in the cytoplasm not surrounded by a cell membrane. Methanogen symbiosis has attracted recent inter- est because methane is a greenhouse gas. Thus, ciliates could potentially contribute indirectly to greenhouse gases by “growing their own meth- ane producers.” Indeed, significant amounts of methane production have been attributed to these ciliate endosymbionts. Up to 95% of the methane production in certain marine habitats has been attributed to the ciliates (Fenchel, 1993), but in other habitats methanogenesis derived from ciliate endosymbionts is a transient and minor contribu- tion (Schwarz & Frenzel,2005). In contrast, over 80% of the methane produced by the American cockroach can be attributed to ciliates (Gijzen & Barugahare, 1992). In other anaerobic habitats, stimulation of bacterial production by ciliate graz- ing can enhance methane production , here not by endosymbiotic bacteria , but by the free-living methanogens . Organic acids, such as acetate and propionate, excreted by the ciliates may stimulate bacterial growth (Biagini, Finlay, & Lloyd, 1998). Research on the endosymbiotic armophoreans , the clevelandellids , has primarily focussed on the symbionts of frogs and insects . The amphibians of Cameroon have provided a rich resource to probe the biology of the clevelandellids . Frog’s eggs are not infected and frog’s with a direct life cycle were never found to carry ciliates. The small frog Phrynodon sandersoni provides a “natural experiment” to confirm these facts. Its tadpoles develop without a digestive tract; of course, the tadpoles are uninfected and so are the adults (Amiet & Affa’a, 1985). Affa’a and coworkers (Affa’a, 1988a; Affa’a & Amiet, 1985, 1994; Amiet & Affa’a, 1985) have concluded that there are three general life histories of infaunation. First, the ciliates may be found only in the juvenile or tadpole stages of the host: this applies to such species as Nyctotheroides brachystomus , Neonyctotherus reticulatus , and Parasicuophora aberrans . Second, other species, such as Nyctotheroides heteros- tomus and Prosicuophora basoglui , infaunate only the adult stage. Finally, both tadpole and adult stages are infaunated by other species, such as Nyctotheroides teochii . We do not know what factors control the dis- appearance of ciliates from the tadpole or the appearance of ciliates in the adults. Affa’a (1986b) has shown that gonadotropins induce encystment in Prosicuophora and Nyctotheroides . It may be that the changes at metamorphosis of the tadpoles induce encystment in those forms that occur only in the tadpole and induce excystment in those forms that occur only in the adult. Ingestion of cysts is probably the main mode of transmission, although infection by live ciliates may occur since the feces of adult frogs have an abundance of ciliates (Amiet & Affa’a, 1985). The prevalence of a ciliate species in a frog host varies from one locality to another, although it is not yet clear what factors determine 8.2 Life History and Ecology 179 180 8. Subphylum 2. INTRAMACRONUCLEATA: Class 2. ARMOPHOREA this variability (Affa’a, 1986a, 1988a). Geographic variation has also been reported for Nyctotherus species that infect cockroaches : similar ciliate genotypes can occur in different insect genera at the same or distant localities (van Hoek et al., 1998). The ciliates apparently have no effect on the amphibian hosts. However, those resident in cockroaches may significantly increase the growth rate and body weight of their hosts (Gijzen & Barugahare, 1992). Reid and John (1983) characterized the cysts of the clevelandellids as flask-shaped, noting similarities to those of the heterotrichs (see also Esteban et al., 1995; Takahashi & Imai, 1989). Cysts are crucial to the maintenance of the life histories of the endo- symbiotic clevelandellids and must certainly be important for those armophorids , such as Metopus , which are found in soils. How widely cyst-forming is distributed in other members of the class remains to be determined. 8.3 Somatic Structures Armophorean ciliates are quite variable in shape and size. Clevelandellids are intermediate in size at around 100 µm; armophorids can range up to 300 µm in length. Shapes are also quite variable. Armophorids , especially caenomorphids , have a rigid, armor-like pellicle with processes and spines , but larger metopids can be quite flexible. The armophorid body is developed into an anterior lobe that can become quite twisted, and along which travel the perizonal or frontal kineties (Fig. 8.1). Smaller forms may have somatic ciliature reduced to anterior and posterior cirrus-like tufts. On the other hand, clevelandellids are very densely ciliated with closely packed somatic kine- ties. These somatic kineties converge on each other forming what are called sutures or secant systems (Fig. 8.1). In clevelandellids , these are typically preoral, apical, caudal, and postoral; the length and precise positions of these secant systems is used in distinguishing genera (e.g., Affa’a, 1983; Albaret & Njiné, 1976; Earl, 1991; Grim, 1998). The cell membrane is underlain by an alveolar layer that may be conspicuous in some caenomor- phids (Fenchel et al., 1977), but it is apparently very compressed, or perhaps even absent, in metopids (Fenchel & Finlay, 1991a) and clevelandellids (de Puytorac & Grain, 1969). Somatic kinetids are dikinetids throughout the class. However, as with the Class SPIROTRICHEA , there is considerable diversity in kinetid structure within the Class ARMOPHOREA . Unfortunately, much of this research remains to be published, appearing only in abstract form or as schematic drawings without micrographic support (Tuffrau & de Puytorac, 1994). We will rely on these but caution that detailed descriptions need to be pub- lished to corroborate the drawings (Fig. 8.2). The armophorid somatic dikinetid is characterized as follows: a ciliated anterior kinetosome with a tan- gential transverse ribbon at triplets 3, 4, 5 and a cili- ated posterior kinetosome with a well-developed divergent postciliary ribbon and a laterally-directed kinetodesmal fibril at triplets 5, 6, 7 that may not be striated (Schrenk & Bardele, 1991). Other micro- tubules have been reported to accompany the ante- rior transverse ribbon near triplets 5 or 6 while a pair of presumably transverse microtubules is situ- ated between the two kinetosomes opposite triplet 4 of the posterior kinetosome (Da Silva Neto in de Puytorac & Tuffrau, 1994; Esteban et al., 1995) (Fig. 8.2). Foissner and Agatha (1999) observed by silver-staining what might be well-developed cathetodesmal fibrils in several Metopus species. The postciliary microtubular ribbons extend along- side each other in the cortical ridges (Fig. 8.3). Paulin (1967) and de Puytorac and Grain (1969) provided the first evidence of the clevelandellid somatic dikinetid of Nyctotherus and Sicuophora , respectively. Grim (1998) has provided some information on the dikinetid of the clevelandellid Paracichlidotherus . The clevelandellid dikinetid can now be characterized as follows: a ciliated anterior kinetosome that bears a tangential trans- verse ribbon at triplets 4, 5 and a striated cathe- todesmal fibril extending to the lateral left from an origin near triplet 2; and a ciliated posterior kinetosome with a divergent postciliary ribbon and a kinetodesmal fibril homologue at triplets 5, 6 (Fig. 8.2). Grim reported two transverse microtu- bules associated with the posterior kinetosome of Paracichlidotherus . The striated cathetodesmal fibrils of clevelandellids may be bifurcated (Fernández- Galiano, 1986; de Puytorac & Grain; de Puytorac & Oktem, 1967). De Puytorac and Grain (1969) illustrated the cathetodesmal fibril of Sicuophora as having two origins, one as indicated above on the anterior kinetosome and the other on the poste- rior kinetosome near the base of the kinetodesmal Fig. 8.2. Schematics of the somatic kinetids of representatives of the Class ARMOPHOREA . ( a ) Dikinetid of Metopus . ( b ) Dikinetid of Paracichlidotherus . ( c ) Dikinetid of Nyctotherus . ( d ) Dikinetid of Sicuophora (from Lynn, 1981, 1991) 8.4 Oral Structures 181 fibril homologue. No micrographic evidence is presented for this interpretation so we have revised our drawing accordingly (Fig. 8.2). We need to havesome detailed reinvestigations of armophoreans before any generalizations can be made about their somatic dikinetids . A further intrigu- ing physiological observation is that Nyctotherus ovalis switches swimming direction in response to voltage changes rather than showing a ciliary reversal. Moreover, this behavior appears to be influenced by host-dependent factors (van Hoek et al., 1999). Contractile vacuoles are present in armopho- reans . The cytoproct is often conspicuous, and in clevelandellids may open to the outside by a cilia- lined channel. Mucocysts appear to be present in the cortex of clevelandellids (Paulin, 1967; de Puytorac & Grain, 1969) and armophorids (Esteban et al., 1995). Finally, mention must be made of the apparent absence of mitochondria with tubular cristae in all armophoreans . The mitochondria in these ciliates have evolved into hydrogenosomes (van Hoek, Akhmanova, Huynen, & Hackstein, 2000a; Boxma et al., 2005). These hydrogenosomes have a hydro- genase that uses electrons derived from pyruvate oxidation to reduce protons and generate hydrogen (Fenchel & Finlay, 1991a; Müller, 1993; Voncken et al., 2002). The hydrogen is typically used in armophoreans by endosymbiotic methanogens (see Life History and Ecology ). 8.4 Oral Structures The armophoreans were placed until recently with the heterotrichs because of their holotrichous somatic ciliation and the presence of multiple oral polykinetids forming an adoral zone . The two or 182 8. Subphylum 2. INTRAMACRONUCLEATA: Class 2. ARMOPHOREA three rows of kinetosomes of the oral polykinetids are hexagonally packed. In armophorids , a third or fourth row of kinetosomes is added continuing the hexagonal packing (Esteban et al., 1995; Foissner & Agatha, 1999). Armophorid oral polykinetids have been called paramembranelles . Clevelandellids typically have three rows of kinetosomes hexago- nally packed, but a fourth, shorter row lies directly opposite to, rather than hexagonally packed with, the kinetosomes of the third row, leading to their designation as heteromembranelles because of the different packing of these kinetosomes of the fourth row (de Puytorac & Grain, 1976). This dif- ferent packing leads to a different orientation and beating of the cilia that was nicely revealed in some published micrographs (Paulin, 1967; Takahashi & Imai, 1989). The adoral zones of armophorids and clevelan- dellids may be quite extensive, spiralling around the body one or more times in some armophorids Fig. 8.3. Somatic cortex of Metopus whose postciliary ribbons extend alongside each other into the cortical ridges. This schema was constructed based on the brief descriptions provided in reports by Schrenk and Bardele (1991) and Esteban et al. (1995) (Fig. 8.1). The clevelandellids have a deeper oral cavity called an infundibulum where the heteromembranelles typically occur (Tuffrau & de Puytorac, 1994). Postciliary ribbons are associ- ated with the kinetosomes of the posterior row in both armophorids and clevelandellids (Tuffrau & de Puytorac). Paroral structures are quite variable in the class. Armophorids appear to have a single file of cilia, which may be derived from linearly arranged oral dikinetids (Esteban et al., 1995; Foissner & Agatha, 1999; Sola, Serrano, Guinea, & Longás, 1992). Clevelandellids have a paroral with two sets of cilia deriving from two files of kinetosomes separated by a ridge (Grim, 1998; Paulin, 1967; de Puytorac & Grain, 1969; Takahashi & Imai, 1989), termed a diplostichomonad by de Puytorac and Grain (1976). The oral structures of armopho- reans are underlain by complex fibrillar structures and microtubules. The filamentous components are implicated in the movement of vesicles to the food vacuole forming region (Eichenlaub-Ritter & Ruthmann, 1983). 8.5 Division and Morphogenesis There have been only a few papers on cell divi- sion and division morphogenesis of armophoreans since Wichterman (1936) described division in Nyctotheroides (= Nyctotherus ). He observed the oral primordium to develop subequatorially. Since silver-staining was not used, kinetosomal replica- tion was not detailed. As far as we know, armo- phoreans divide while swimming freely. Foissner (1996b) has characterized stomatogenesis as pleu- rotelokinetal (i.e., occurring within or at the end of several somatic kineties). Two studies on the armophorids , Metopus and Caenomorpha , demonstrated pleurotelokinetal stomatogenesis . Martín-González, Serrano, and Fernández-Galiano (1987) showed that the oral primordium in Caenomorpha develops by prolif- eration from the posterior ends of many perizonal somatic kineties . The primordial field splits later in development with an anterior portion devel- oping into the paroral and the posterior portion developing into the oral polykinetids . In Metopus , a number of posterior dorsolateral somatic kineties begin to proliferate kinetosomes (Foissner & Agatha, 1999). These differentiate as the oral polykinetids (Fig. 8.4). The paroral differenti- ates later. Foissner and Agatha (1999) interpreted it to develop from kinetosomes derived from perizonal kineties . However, it is just as pos- sible from the evidence presented that paroral dikinetids could derive from “anterior” or “right- side” kinetosomes in a fashion very similar to that reported for Caenomorpha . If this were the case, there would be strong similarities in Fig. 8.4. Division morphogenesis of Metopus , a representative of the Class ARMOPHOREA . ( a ) Kinetosomal replication begins at the “equatorial ends” of a number of somatic kineties. ( b ) Oral polykinetids assemble through side-by-side alignment of dikinetids units. ( c ) The posterior ends of several somatic kineties adjacent to the develop- ing oral region disassemble, and it may be that the paroral ( d, e ) is assembled from these as division proceeds. (from Foissner & Agatha, 1999.) 8.5 Division and Morphogenesis 183 184 8. Subphylum 2. INTRAMACRONUCLEATA: Class 2. ARMOPHOREA stomato genesis between these two genera. Caenomorpha undergoes a complicated post-sto- matogenesis morphogenesis, reminiscent of the enantiotropic division of some oligotrichous spiro- trichs (Martín-González et al., 1987). Considering the current evidence, we are not convinced that the differences between metopids and caenomorphids are sufficient to justify ordinal status for these two groups, as suggested by Foissner and Agatha (1999). Santos, Guinea, and Fernández-Galiano (1986) have provided a preliminary account of stoma- togenesis in the clevelandellid Nyctotherus . Breaks occur in somatic kineties posterior to the oral region and kinetosomal proliferation occurs at the anterior ends of these breaks. A lateral groove develops as proliferation proceeds and primordium elements on the posterior wall of the groove differentiate as oral polykinetids while those on the anterior wall develop as paroral dikinetids, eventually forming the two files of the diplostichomonad (Santos et al., 1986). This is clearly a pleurotelokinetal stomato- genesis , showing significant similarities to that of the armophorids. 8.6 Nuclei, Sexuality and Life Cycle Armophoreans have the typical complement of macronucleus and one or more micronuclei . The macronuclei can also be variable in number in caenomorphids , sometimes numbering more than four (Fig. 8.1). In smaller forms, the macronucleus is typically globular to ellipsoid, but in larger clevelandellids it can become elongated and quite irregular in shape. The macronucleus of some clevelandellids is “suspended” from the cortex by microfibrillar strands that collectively are called the karyophore (Fig. 8.1). Eichenlaub-Ritter and collaborators have under- taken some detailedultrastructural studies on micronuclear and macronuclear division in the clevelandellid Nyctotherus cordiformis . Macronuclei divide by intramacronuclear microtubules that are primarily responsible for the elongation of the macronucleus, which is also accompanied on its out- side by scattered extramacronuclear microtubules (Eichenlaub-Ritter & Tucker, 1984; Hamelmann, Eichenlaub-Ritter, & Ruthmann, 1986). Micronuclear mitosis is an endomitosis, typical of ciliates (Raikov, 1982). There may be three “classes” of micro tubules, identified by their differing responses to drugs and temperature, which function to accomplish micronu- clear mitosis : (1) manchette microtubules underlying the nuclear envelope; (2) interpolar and kinetochore microtubules, which function during anaphase; and (3) stembody microtubules, which function during telophase to separate the putative micronuclei to each progeny cell (Eichenlaub-Ritter & Ruthmann, 1982a, 1982b). Microtubules in the dividing nuclei may have more than the canonical 13-protofila- ments (Eichenlaub-Ritter, 1985; Eichenlaub-Ritter & Tucker). Conjugation has been studied in only a few examples of armophoreans since the description of it in Nyctotheroides (= Nyctotherus ) by Wichterman (1936). It is not established what factors stimulate conjugation in free-living forms. Wichterman (1936) observed it occurring only in transforming tadpoles of the frog Hyla versicolor . This lead to specula- tion that gonadotropins or some other physiological signal derived from the host may cue these ciliates to begin conjugation. However, Sandon (1941a) observed conjugation in Paranyctotherus isolated from the adult clawed frog Xenopus laevis , sug- gesting that other factors are involved. Affa’a and Amiet (1994) have confirmed that conjugation can occur in all stages of the frog life cycle – tadpoles , transforming individuals, and adults. Gonadotropin injections induced conjugation in Prosicuophora , even when immature stages were treated (Affa’a, 1986b). Thus, it is unlikely that one single factor stimulates conjugation . Fusion of the conjugants occurs in the anterior region, and in some Metopus species total conjuga- tion may occur (Noland, 1927). The micronuclei of each partner typically undergo three maturation divisions – two meiotic divisions followed by a mitosis of one of the four haploid products (Raikov, 1972; Martín-González et al., 1987). In the total conjugation of Metopus , the cytoplasm of one con- jugant flows into the partner carrying the gametic nucleus or nuclei with it. However, the old macro- nucleus is left in the cortical shell of the disgarded partner (Noland, 1927). Following fusion of the gametic nuclei to form the synkaryon , armopho- reans typically have one post-synkaryon division with one nucleus becoming the new micronucleus and the other becoming the new macronucleus. In species with more than one macronucleus, there may be additional post-synkaryon divisions (see Martín-González et al., 1987). Development of the macronuclear anlage in armophoreans is an extremely long process: Golikova (1965) recorded it taking up to 2 weeks in Nyctotheroides (= Nyctotherus ) while Noland (1927) observed a mininimum of 1 week in Metopus . In both these genera, it appears that polytene chromo- somes are formed at one stage during anlage devel- opment. Golikova (1965) concluded that one giant polytene chromosome may form in Nyctotheroides by the end-to-end joining of the individual chromo- somes. This giant chromosome later fragments both transversely and longitudinally to yield the macro- nuclear chromosomes (Vinnikova & Golikova, 1978). Ultimately, the macronuclear chromosomes fragment into gene-sized pieces as happens in the Class SPIROTRICHEA (see Chapter 7 ), a fact that Riley and Katz (2001) have confirmed by molecular analyses of the macronuclear DNA of both armophorids and clevelandellids. 8.7 Other Features The free-living armophorids have been recog- nized for some time as strong indicators of anoxic aquatic environments (e.g., Bick, 1972; Foissner, 1988a; Sládecˇek, 1973). They are commonly found in soils (Foissner, 1987) and have been recorded from a variety of municipal landfill sites in the United Kingdom, where they undergo an encyst- ment - excystment cycle in response to starvation and water loss (Finlay & Fenchel, 1991). 8.7 Other Features 185
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