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129 Abstract Ciliates in this class were thought to represent the pinnacle of ciliate evolution, along with the spirotrichs. However, small subunit rRNA gene sequences and the presence of postciliodes- mata in the somatic cortex strongly relate members of this class to the Class KARYORELICTEA. The heterotrichs are typically majestic ciliates of large cell size and with a conspicuous adoral zone of polykinetids or membranelles (AZM) that extend out over the peristomial surface. The ciliates in this class are not subdivided, and so there is one order – Order Heterotrichida. Heterotrichs are found in a diversity of habitats, from the marine benthos and hydrothermal vents to the plankton of high altitude oligotrophic lakes. They feed on a diversity of prey, ranging from bacteria up to small metazoa, like rotifers, and sometimes are conspicuous by carry- ing symbiotic zoochlorellae. Their body is highly contractile, elongated by postciliodesmal micro- tubules and shortened by contractile myonemes. The oral structures have a paroral and multiple paramembranelles. Stomatogenesis is parakinetal. Macronuclei can be nodular, and are divided by extramacronuclear microtubules. Conjugation has not been studied in any breadth in the class with the gamone-receptor system of Blepharisma being the only model. The heterotrich Spirostomum has been developed as a bioassay model for heavy metal. Keywords Ampliploid, microbiotest Heterotrichs are common and large ciliates, some Spirostomum species achieving body lengths of up to 4,000 µm. They include some of the best-known and most common ciliates in the phylum. Stentor , a typical representative, has long attracted attention from protozoologists and cell biologists because of its size, ubiquity, ease of general laboratory culture, contractility, and regenerative powers (Fig. 6.1). Typically heterotrichs are free-swimming and holot- richously ciliated, although members of the Family Folliculinidae secrete attached loricas in which they live. The group is at least 100–200 million years old as demonstrated by the fossil ized lorica of Priscofolliculina (Deflandre & Deunff, 1957). Heterotrichs were so-named because of the marked difference between their holotrichous somatic ciliation and the conspicuous, typically spiralling adoral zone of membranelles or oral polykinetids . They were conceived as the pivotal group in the evolution of the “higher” or polyhy- menophorean ciliates (Corliss, 1961, 1979). Doubt about this vision began to emerge in the late 1970s. Ultrastructural data indicated dramatic differences in their somatic kinetids compared to other polyhy- menophoreans ; and similarities in the heterotrich somatic cortex to that of the karyorelicteans sug- gested a closer relationship between the presuma- bly most derived and the presumably most ancestral groups (Gerassimova & Seravin, 1976; Lynn, 1981, 1991). The nature of membrane particle arrays in different ciliate groups also suggested a stronger relationship between heterotrichs and karyorelict- eans (Bardele, 1981). Finally, sequences of nuclear ribosomal RNA genes from heterotrichs and karyorelicteans supported their sister group status in an early diverging lineage (Baroin-Tourancheau, Delgado, Perasso, & Adoutte, 1992; Greenwood, Schlegel, Sogin, & Lynn, 1991b; Hirt et al., 1995). Chapter 6 Subphylum 1. POSTCILIODESMATOPHORA: Class 2. HETEROTRICHEA – Once Close to the Top 130 6. Subphylum 1. POSTCILIODESMATOPHORA: Class 2. HETEROTRICHEA – Once Close to the Top Fig. 6.1. Representative genera of the Class HETEROTRICHEA . Blepharisma with a somewhat linear arrangement of the adoral zone of polykinetids along the left margin of the oral region. In contrast, the oral polykinetids of Stentor and Climacostomum spiral out of the oral cavity in a counter-clockwise direction, bounding a peristomial field that is covered by kineties. The folliculinid Eufolliculina exemplifies this unique family of heterotrichs in being anchored in a lorica and in having its oral region drawn out into two extensive peristomial “wings” Thus, we are now certain that the postciliodesmata , shared by both karyorelicteans and heterotrichs , demonstrate their shared common ancestry. The ciliates now assigned to this class are united by four major features. First, they have highly ampliploid and dividing macronuclei. Macronuclear karyokinesis is accomplished primarily by extramacronuclear microtubules (Diener, Burchill, & Burton, 1983; Jenkins, 1973; Lynn & Small, 1997) and probably evolved inde- pendently of macronuclear karyokinesis in the Subphylum Intramacronucleata (Lynn, 1996a; Orias, 1991a; and see Chapter 4 ). Second, the microtubular components of their postciliodes- mata are more simply organized than those of the karyorelicteans : they appear as ribbons oriented perpendicular to the cell surface, only separated by a single microtubule. Third, the oral polyki- netids on the left side of the oral region are char- acterized as paramembranelles (de Puytorac & Grain, 1976), which form a conspicuous adoral zone often extending out onto the anterior cell surface. Fourth, the differentiation of these oral polykinetids during stomatogenesis occurs from the center of the oral primordium towards the anterior and posterior, a unique pattern within the phylum (Aescht & Foissner, 1998). 6.1 Taxonomic Structure There has been considerable change in the composition of this taxon since Corliss (1961, 1979). Corliss (1979) recognized six suborders within the Order Heterotrichida : (1) Suborder Heterotrichina ; (2) Suborder Clevelandellina ; (3) Suborder Armophorina ; (4) Suborder Coliphorina ; (5) Suborder Plagiotomina ; and (6) Suborder Licnophorina . Results from the study of ultrastructure and molecular sequences now suggest the following. The somatic kinetids of clevelandellids (Affa’a, unpublished, 2007; de Puytorac & Grain, 1969), armophorids (Schrenk & Bardele, 1991), plagioto- mids (Albaret & Grain, 1973), and licnophorids (Da Silva Neto, 1994a) do not exhibit postciliodesmata and also have different patterns of fibrillar associ- ates (Lynn, 1981, 1991). Furthermore, nuclear small subunit rRNA (SSrRNA) gene sequences separate the clevelandellids (van Hoek, van Alen, Sprakel, Hackstein, & Vogels, 1998, 2000b) and armo- phorids (Embley, Finlay, Thomas, & Dyal, 1992; van Hoek et al., 1998, 2000b) to a new class, the Class ARMOPHOREA (see Chapter 8 ), while plagiotomids (Affa’a, Hickey, Strüder-Kypke, & Lynn, 2004) and licnophorids (Lynn & Strüder-Kypke, 2002) are transferred to the Class SPIROTRICHEA (see Chapter 7 for details). The Suborder Coliphorina only included the Family Folliculinidae . However, the somatic kinetids of Eufolliculina are extremely similar to other heterotrichs in their fibrillar associates and the character of the postciliodesmata (Mulisch, Barthlott, & Hausmann, 1981), while SSrRNA sequences indicate this genus falls within the heter- otrich radiation and is the sister taxon to Maristentor (Miao, Simpson, Fu, & Lobban, 2005). Thus, this group should not be separated at such high rank, and we do not now recognize this suborder. Within the Suborder Heterotrichina , Corliss (1979) included the following: Family Bursariidae , Family Chattonidiidae , Family Climacostomidae , Family Condylostomatidae , Family Metopidae , Family Peritromidae , Family Phacodiniidae , Family Reichenowellidae , Family Spirostomidae , and Family Stentoridae . The somatic kinetids of Phacodinium (Didier & Dragesco, 1979; Da Silva Neto, 1993a), Transitella , a reichenowellid (Foissner, Adam, & Foissner, 1982; Iftode, Fryd-Versavel, Wicklow, & Tuffrau, 1983), metopids (Schrenk & Bardele, 1991), and bursariids (Gerassimova, Sergejeva, & Seravin,1979; Lynn, 1980) do not form postcili- odesmata , while redescriptions of the reichenowel- lid Balantidioides suggest that it has affinities to the spirotrichs (Foissner et al., 1982). While SSrRNA gene sequences support placement of Phacodinium among the spirotrichs (Shin et al., 2000), these same gene sequences confirm the heterotrich affinities of Peritromus (Rosati, Modeo, Melai, Petroni, & Verni, 2004), Chattonidium , (Modeo et al., 2006), and Condylostomides (Schmidt, Foissner, Schlegel, & Bernhard, 2007). In conclusion, we now recognize one order within the class, the Order Heterotrichida with characters of the class, and eight families: Family Blepharismidae [but see Aescht & Foissner, 1998], Family Chattonidiidae Family Climacostomidae , Family Condylostomatidae , Family Maristentoridae , Family Peritromidae , Family Spirostomidae , and Family Stentoridae (see Chapter 17. Ciliate Taxa ). 6.1 Taxonomic Structure 131 132 6. Subphylum 1. POSTCILIODESMATOPHORA: Class 2. HETEROTRICHEA – Once Close to the Top They are distinguished primarily by features of the oral region and variations in their overall body form. A number of works have treated different genera in detail: Spirostomum (Repak & Isquith, 1974); Blepharisma (Repak, Isquith, & Nabel, 1977); Stentor (Foissner & Wölfl, 1994); and a recent report on the rare genus Copemetopus (Al-Rasheid, 2001). We should not forget the classic works on Stentor , the majestic “king of the ciliates”, by Tartar (1961) and on Blepharisma , the light-sensitive pro- tozoon by Giese and collaborators (1973). Hadži (1951) is the classic work on the folliculinids . 6.2 Life History and Ecology Because of their typically large size, heterotrichs can be conspicuous members of microbial foodwebs and have a widespread distribution. Heterotrichs have been recorded from freshwater lakes in subtropical Florida (Beaver & Crisman, 1989b), Antarctica (Kepner, Wharton, & Coats, 1999), Europe (Finlay, 1982), and high altitude lakes in South America (Woelfl & Geller, 2002), and streams in Europe (Madoni & Ghetti, 1980). They are found in a variety of marine habi- tats, including anaerobic sediments in Europe (Fenchel & Finlay, 1990a), the marine sublittoral in Europe (Agamaliev, 1971; Azovsky & Mazei, 2003; Kovaleva & Golemansky, 1979; Mazei & Burkovsky, 2003) and even deep marine habitats (Fenchel et al., 1995) and hydrothermal vents (Small & Gross, 1985; Bergquist et al., 2007). Heterotrichs are often dominant members of the low diversity ciliate communities of hypersaline habitats across the globe – in Europe (Esteban & Finlay, 2004), Africa (Yasindi, Lynn, & Taylor, 2002), Arabia (Al-Rasheid, Nilsson, & Larsen, 2001; Elloumi et al., 2006), and Australia (Post, Borowitzka, Borowitzka, Mackay, & Moulton, 1983). They are occasionally found in soils (Buitkamp, 1977; Foissner, 1998a; Griffiths, 2002). Most species are free-swimming, but some, such as Stentor , have the ability to use a holdfast to temporarily attach to the substrate (Fauré- Fremiet, 1984). A few species of Stentor secrete a mucoid sheath and all species of folliculinids secrete a lorica in which they can retract to avoid predation. The substances for these external coverings originate from extrusomes (Bussers, 1984; Mulisch & Hausmann, 1983), and in the folliculinids may contain chitin fibrils (Mulisch, Herth, Zugenmaier, & Hausmann, 1983). Substrates to which heterotrichs attach include inorganic substrates and macrophytes. Folliculinids attach to the integument of various invertebrates (Matthews, 1968; Fernández-Leborans & Córdoba, 1997), and may cause the skeletal eroding band or brown band diseases of scleractinian corals (Antonius, 1999; Cróquer et al., 2006). Maristentor is found on corals, but does not appear to cause disease (Lobban et al., 2002). Some genera, like Fabrea , are strictly marine or brackish water forms, which can attain abundances of 10 5 l −1 (Elloumi et al., 2006; García & Niell, 1993). Stentor species can reach more than 10 3 l −1 in some lakes in the southern hemisphere, perhaps due to the absence of larger microcrustacean predators (James, Burns, & Forsyth, 1995; Laybourn-Parry, Perriss, Seaton, & Rohozinski, 1997). Dispersal generally occurs by swimming, but cysts may also be involved (see below). Kusch (1998) has demon- strated clear evidence of relatively high gene flow among populations of Stentor separated by as much as 400 km. Genera in the Family Folliculinidae are typically marine although the fresh-water species Folliculina boltoni has been recorded from Europe (Penard, 1919), North America (Hamilton, 1952), and South America (Dioni, 1972) while Ascobius lentus has been recorded recently in European freshwaters (Mulisch, Heep, Sturm, & Borcherding, 1998). Folliculinids are dispersed in part by the movements of their host, but the proter or anterior daughter differentiates at cell division as a “mouthless swarmer ” stage that is adapted for dispersal. Heterotrichs are omnivorous, upstream filter feed- ers (Fenchel, 1980a), showing little preference for prey species. Bacteria , autotrophic and heterotrophic flagellates , and ciliates are ingested, with some prey species proving more nutritious than others (Rapport, Berger, & Reid, 1972; Repak, 1983, 1986). Heterotrichs may change the shape of the oral region (Liebsch, 1976) and the spacing between the cilia of the oral polykinetids (Rickards & Lynn, 1985) in response to physiological states and prey types. When smaller food items become scarce, heterot- richs can become cannibalistic (Foissner & Wölfl, 1994; Giese, 1973; Pierce, Isquith, & Repak, 1978) and have also been known to ingest smaller metazoans (Foissner & Wölfl; Tartar, 1961). In an unusual turn of the tables, it appears that Mirofolliculina limnoriae , an epibiont on the wood-boring isopods of the genus Limnoria , may outcompete its host for food and hinder host dispersal, suggesting it can be considered an ectoparasite (Delgery, Cragg, Busch, & Morgan, 2006). Heterotrichs harbor a variety of endosymbionts : bacteria can be found in the cytoplasm and in the macronucleus (Fokin, Schweikert, Brummer, & Görtz, 2005; Görtz, 1983; Görtz & Wiemann, 1987). The bacterial endosymbionts do not appear to be harmful; in fact, some bacteria may be essential sym- bionts (Hufschmid, 1984). A variety of Chlorella spe- cies provide their Stentor and Climacostomum hosts with the “by-products” of photosynthesis (Fernández- Leborans & Zaldumbide, 1983; Kawakami, 1984; Reisser, 1984; Woelfl & Geller, 2002), and may com- pete with bacterial endosymbionts for the host cyto- plasmic niche (Hufschmid, 1984). Laybourn-Parry et al. (1997) determined that Stentor amethystinus could contribute almost 70% of the total plankton photosyn- thesis in some Australian lakes. Heterotrichs themselves are prey for other ciliates and metazoans. Stentor has mechanoreceptors dis- tributed on its cell surface that may enable response to predator contact (Wood, 1989). When contact is made with toxicyst-bearing litostome ciliates, like Dileptus (see Chapter 9 ), Blepharisma (Harumoto et al., 1998; Miyake, Harumoto, Salvi, & Rivola, 1990), Climacostomum (Masaki et al., 1999), and Stentor (Miyake, Harumoto, & Iio, 2001) induce a massive release of their pigmentocysts , respectively containing the pigments blepharismin , climacostol , and stentorin , which have proved lethal to this predator. However, the pigment does not inhibit predation by the heterotrich Climacostomum on its heterotrich relative Blepharisma (Terazima & Harumoto, 2004). Pigmented heterotrichs also exhibit light-sensitive behavior (Giese, 1973). Their photophobic response appears as a ciliaryreversal when the intensity of incident light suddenly increases. The mechanism is likely due to a release of H + by the pigment. These ions are then translocated to the cytoplasm, causing electrical potential changes in the plasma membrane and subsequent ciliary reversal and reorientation of the cell (Fabczak, Fabczak, & Song, 1993a; Fabczak et al., 1993b; Matsuoka & Kotsuki, 2001; Menzies, Das, & Wood, 2004; Sobierajska, Fabczak, & Fabczak, 2006), perhaps involving a G-protein-mediated sig- nalling pathway (Fabczak, Sobierajska, & Fabczak, 2004). Certainly, a photophobic response might be a selective advantage, keeping the ciliate hidden from potential predators. However, one cannot help but wonder which trait is under selection: the photopho- bic response mediated by the pigment chemicals or the toxic nature of the pigment chemicals themselves (Lobban, Hallam, Mukherjee, & Petrich, 2007). Heterotrichs can survive several weeks without food (Jackson & Berger, 1985a, 1985b). Nevertheless, encystment is a common feature of this class, stimu- lated by a variety of factors, such as absence of food or excess metabolites (Giese, 1973; Repak, 1968). The cyst wall is formed of several layers, which may contain chitin (Mulisch & Hausmann, 1989). Excystment occurs through a cyst pore plug or micropyle . It may be induced by freshly bacterized medium (Giese, 1973; Repak, 1968), and is perhaps promoted by a substance liberated from excysting conspecifics (Demar-Gervais & Génermont, 1971). Conjugating Stentor have been observed rarely in nature (Burchill, George, Lindberg, & Sims, 1974; Tartar, 1961). Stentor coeruleus mates with some frequency in the laboratory, perhaps induced by elevated temperatures (Rapport, Rapport, Berger, & Kupers, 1976), while Blepharisma has served as a model for understanding heterotrich sexual processes (Miyake, 1996). The marine heterotrich Fabrea requires a complex organic medium to complete conjugation (Demar−Gervais, 1971). 6.3 Somatic Structures The heterotrich cell body is quite variable in shape (Fig. 6.1) depending upon whether the ciliate is a benthic or substrate oriented species or a more planktonic species. The body is covered by numer- ous bipolar somatic kineties composed of dikinetids (Tuffrau, 1968). There is a fine glycocalyx on top of the plasma membrane, which is underlain by an alveolar layer that is often not well-developed and appears to be discontinuous. The epiplasm is very thin and inconspicuous. Mucocysts and pig- mentocysts are found beneath the alveolar layer, giving the range of “living colors” in this group – black, blue, blue-green, brown, rose, and yellow. Chlorella symbionts may impart a grass-green color to those species harboring them. 6.3 Somatic Structure 133 134 6. Subphylum 1. POSTCILIODESMATOPHORA: Class 2. HETEROTRICHEA – Once Close to the Top The somatic kinetids are typically dikinetids that lie 20–40° to the long axis of the kinety (Grain, 1984; Lynn, 1981, 1991; Fabrea – Da Silva Neto & Grolière, 1993; Condylostomides – Da Silva Neto, 1994b). The anterior kinetosome is often the only one ciliated and it bears a tangential transverse ribbon of about 6 microtubules near triplets 3, 4, and 5. This ribbon is usually doubled by a single microtubule on the right inside edge (Fig. 6.2). The posterior kinetosome is less often ciliated and may have a transverse ribbon associated with it, oriented in a variety of ways. There is a kineto- desmal fibril homologue originating near triplets 5, 6, which extends laterally, usually associating with the postciliary ribbon originating from the next anterior dikinetid. It is called a homologue because it usually does not have the obvious periodic striation found in other ciliates, although it arises from the same triplet region. The post- ciliary ribbon of this kinetosome is divergent and extremely well-developed, numbering 12 or more microtubules, which extend towards the cortex as ribbons oriented perpendicular to the cell surface and separated by a single microtubule (Fig. 6.2). These postciliary ribbons are accompanied by dense material called a retrodesmal fibril (Grain, 1984) or a postciliary accessory fibre (Peck, Pelvat, Bolivar, & Haller, 1975). Yogosawa-Ohara, Suzaki, and Shigenaka (1985) suggest that this fibre may induce the twisting of the body during contractions of Spirostomum . A number of postciliary ribbons are integrated together to form the conspicuous postciliodesma or Km fiber in the cortex of these ciliates. Myonemes are typically present and always so in contractile forms. They are predominantly longi- tudinally arranged around the entire body, and are either in direct contact with the somatic kinetosomes or indirectly contact them via intermediate fibres. Transverse myonemes may integrate the longitudi- nal bundles, either locally or throughout the cortex. These cortical contractile systems created consider- able interest among cell biologists who sought to explain their role in changing cell shape. Huang and Pitelka (1973) first experimentally demonstrated the antagonistic relationship between the myonemes and postciliodesmata in Stentor : the myonemes are responsible for contraction of the body while micro- tubule-on-microtubule sliding achieves the slow elon- gation back to the “relaxed” form. This system has now been demonstrated in normal contractions of Spirostomum (Yogosawa-Ohara & Shigenaka, 1985; Yogosawa-Ohara et al., 1985) and in the light- induced contractions of the posterior portion of the body in Blepharisma (Ishida, Suzaki, & Shigenaka, 1991a; Ishida Suzaki, Shigenaka, & Sugiyama, 1992). Contraction and elongation involve calcium (Ishida et al., 1992; Legrand, 1971), which may be stored as hydroxyapatite in crystalline intracellular deposits (Takagui & Silveira, 1999) or in cortical alveoli (Ishida, Shigenaka, Suzaki, & Sugiyama, 1991b). However, there are conflicting reports regard- ing the inhibitory action of cytochalasin , an actin antagonist, on contraction (Ettienne & Selitsky, 1974; Yogosawa-Ohara & Shigenaka). Although we can- not yet conclude that this is an actin-based system, a caltractin-like protein has been localized to the myonemes of Stentor (Maloney, McDaniel, Locknar, & Torlina, 2005). The contractile vacuole system is well- developed in heterotrichs , especially the fresh-water forms, which may have conspicuous collecting canals (Patterson, 1976, 1980). Mucocysts are probably widespread among heterotrichs, since they often encyst . However, there are relatively few studies on these. Mulisch and Hausmann (1983) documented their role in lorica construction in folliculinids . Pigmentocysts are considered to be a special type of mucocyst (Hausmann, 1978). 6.4 Oral Structures Tuffrau (1968) presented a detailed description of the heterotrich oral region and its fibrillar supports, but this diversity has been considerably reduced with the removal of a number of groups from this class (see Taxonomic Structure above). The oral region is characterized by an adoral zone of polyki- netids , which typically form a small dextral spiral around the cytostomal region deeper in the oral cavity. The oral polykinetids then extend out of this deeper cavity onto the cell surface of the oral region where variations in the pattern become more conspicuous: they may be organized as a linear file along the left border; may form a more or less com- plete circle around the anterior end, or may extend out on two wing-like projections in the folliculinids (Fig. 6.1). The peristomial region circumscribed by 6.4 Oral Structures 135 Fig. 6.2. Ultrastructure of the cortex of the Class HETEROTRICHEA . A Somatic dikinetids . ( a ) Blepharisma (after Ishida et al., 1991a). ( b ) Climacostomum (after Peck et al., 1975). ( c ) Eufolliculina(after Mulisch et al., 1981). Note the single transverse microtubule at the right end of the transverse ribbon (T) of the anterior kinetosome and the variable arrangement of transverse microtubules (T) associated with the posterior kinetosome. Kd, kinetodesmal fibril homologue ; Pc, postciliary microtubular ribbon . B Somatic cortex of Blepharisma with postciliodesmata composed of overlapping ribbons in the ribbon + 1 arrangement. (Redrawn after Ishida et al., 1992.) 136 6. Subphylum 1. POSTCILIODESMATOPHORA: Class 2. HETEROTRICHEA – Once Close to the Top the oral polykinetids may be covered by somatic kineties in some forms (e.g., Stentor ) or not in oth- ers (e.g., Condylostoma ). The oral polykinetids , termed paramembranelles (de Puytorac & Grain, 1976), are minimally composed of a row of dikinetids, whose anterior kinetosomes bear a transverse ribbon and poste- rior kinetosomes bear a divergent postciliary rib- bon. Additional complete or incomplete rows of kinetosomes may be added to these first two “rows”, depending upon the species and upon the position in the adoral zone at which the oral polykinetid lies. Kinetosomes on the right border of the second and third rows may also have postciliary micro tubules. For example, in Stentor , those polykinetids furthest from the cytostome have only two to four kinetosomes in the third row while closer to the cytostome this number increases to twenty, close to the number of kinetosomes in the first two rows (Jacobson & Lynn, 1992). This is also the case for other genera (Mulisch & Hausmann, 1984; de Puytorac & Grain, 1976; Da Silva Neto, 1993b, 1994b). However, the number of kinetosomes in each oral polykinetid may be reduced as the cell size of the heterotrich decreases (Jacobson & Lynn, 1992). It is in the structure of the paroral that most variation is seen. This is presumably because the conservation of the paroral structure is less critical to feeding function in upstream filter feeders like the heterotrichs . De Puytorac and Grain (1976) have provided definitions for a variety of terms applied to describe the structure of the heterotrich paroral (see also the Glossary ; Mulisch & Hausmann, 1984; Da Silva Neto, 1994b). These include paro- ral in pairs (e.g., Climacostomum ), stichodyad (e.g., Blepharisma , Stentor ), stichomonad (e.g., Spirostomum ), and polystichomonad (e.g., Condylostoma ). Our knowledge of this diversity is increased with each new description (e.g., Fabrea , Da Silva Neto, 1993b; Condylostomides , Da Silva Neto, 1994b). It is further complicated by vari- ations within the same species along the course of the paroral from near the cytostome to out onto the body surface (e.g., Stentor , Bernard & Bohatier, 1981; Climacostomum , Fischer-Defoy & Hausmann, 1981; Condylostomides , Da Silva Neto, 1994b). We conclude that it is futile to capture this diversity by establishing new terms for each new paroral structure described, and we recommend that descriptions preface the character of the paro- ral with the taxonomic name of the group (e.g., Stentor paroral). Tuffrau (1968) diagrammed the variety of fibril- lar structures that support the cell surface of the oral region and the cytopharynx. Ultrastructural studies have demonstrated these to be kinetosomal postciliary ribbons and nematodesmata that extend from the bases of the oral polykinetids and paroral kinetosomes (Grain, 1984). The nematodesmata from adjacent polykinetids join to form overlapping microtubular rootlets, which may serve a cytoskeletal function for the oral region and may also be involved in re-extension of the oral region, especially in folliculinids with their elongated peristomial wings (Mulisch & Hausmann, 1984). The cytopharynx may be supported by paroral postciliary microtu- bules ( Climacostomum , Fischer-Defoy & Hausmann, 1981) or oral polykinetid postciliary microtubules (Eufolliculina , Mulisch & Hausmann, 1984). Filamentous structures are often observed in the cytostomal region. As for other ciliates, Mulisch and Hausmann (1984) have proposed that these function to facilitate the pinching off of the forming food vac- uole. This hypothesis has received support in experi- ments using cytochalasins , “anti-actin” drugs, which inhibit phagocytosis in Spirostomum (Zackroff & Hufnagel, 1998). Once food is ingested, food vacu- oles and primary lysosomes fuse, and eventually the food vacuole fragments into smaller vesicles, which may distribute digesting materials (Fischer- Defoy & Hausmann, 1982). The defecation vacuole ultimately fuses with the plasma membrane at the cytoproct and its membrane is presumably recycled by the cell (Fischer-Defoy & Hausmann, 1992). 6.5 Division and Morphogenesis Heterotrichs typically divide while swimming freely, and are characterized as having parakinetal stomatogenesis (Foissner, 1996b). The early studies of Fauré-Fremiet (1932) and Villeneuve-Brachon (1940) demonstrated that kinetosomal replication in one to several subequatorial kineties produced the initial anarchic field of the oral primordium. This often occurs in the zone of stripe contrast, a region where there is a marked contrast in the width of the pigment stripes or interkinetal separation (Frankel, 1989; Tartar, 1961). This anarchic field may be in the ventral region in the long axis of the body posterior to the proter oral region (e.g., Blepharisma ) (Fig. 6.3) or might even occur on the dor- sal surface (e.g., Fabrea ). Initially, the anarchic field is composed of unoriented kinetosomal pairs (Bernard & Bohatier, 1981; Mulisch & Hausmann, 1988). Eventually, the anarchic field divides lon- gitudinally and paroral structures differentiate on its right side while adoral structures differentiate on its left side. Oral polykinetids initially differ- entiate in the centre of the oral primordium as dikinetids assemble from the right towards the left. Additional oral polykinetids join these central ones Fig. 6.3. Stomatogenesis of the heterotrich Blepharisma . ( a ) The process begins with proliferation of kinetosomes and dikinetids along a ventral postoral kinety. ( b ) The oral polykinetids begin to differentiate as dikinetids align beginning in the middle of the anlage and extending towards each end. ( c ) Differentiation continues towards the ends, visible at this stage by the addition of a third row to oral polykinetids initially in the middle of the anlage. The paroral begins to differentiate in the posterior right region ( d ) The paroral continues its differentiation as the adoral zone begins to curve towards and right in preparation for invagination of the opisthe’s oral cavity. Note that there is some dedifferentiation and redifferentiation of the oral structures of the proter. (From Aescht & Foissner, 1998.) 6.5 Division and Morphogenesis 137 138 6. Subphylum 1. POSTCILIODESMATOPHORA: Class 2. HETEROTRICHEA – Once Close to the Top as the adoral field develops from the center towards the two ends of the primordium (Fig. 6.3). This latter feature is considered a strong apomorphy for the class (Aescht & Foissner, 1998; see also Shao et al., 2006). Foissner (1996b) has described and defined the variations in heterotrich stomatogenesis: it is parakinetal when one kinety is involved, polypara- kinetal when more than one kinety is involved, and amphiparakinetal when the oral primordium curves to intersect somatic kineties at both its anterior and posterior ends. These intersected somatic kineties become the peristomial field kineties in Stentor and Fabrea , but are resorbed in folliculinids. The parental or proter oral apparatus may be only slightly dedifferentiated during cell division (e.g., Blepharisma , Condylostoma , Stentor ) or entirely dedifferentiated and regenerated (e.g., Spirostomum ). In folliculinids, the entire proteroral apparatus is dedifferentiated as the proter becomes the dispersal “ swarmer ” stage, which has a very reduced spiral of membranelles and does not form food vacuoles. Differentiation of the proter oral apparatus occurs upon settling and shows a similar pattern to the development of the opisthe oral apparatus prior to cell division (Mulisch & Hausmann, 1988). Heterotrichs, like the karyorelicteans, have remarkable regenerative abilities that have long been exploited to probe how the development of cell pattern is regulated at the morphological (e.g., De Terra, 1985; Fahrni, 1985; Tartar, 1961; Uhlig, 1960) and biochemical levels (Bohatier, 1981, 1995), a subject area that is well beyond the scope of our treatment here, but see Frankel (1989) for a comprehensive review. 6.6 Nuclei, Sexuality and Life Cycle The macronuclei of heterotrichs range from compact ellipsoid to ribbon-like and finally to moniliform or beaded. Correlated with their large cell size, heterotrich macronuclei are large and highly amplified. Nucleoli are often promi- nent. In Stentor , the nuclear envelopes of both macronucleus and micronuclei are surrounded by an additional membrane system that integrates them structurally to the endoplasmic reticulum (Mulisch, 1988). Since Mulisch (1988) used special fixation techniques to obtain this result, it may be a more general property of heterotrichs than is presently realized. Heterotrichs typi- cally have many micronuclei distributed along the length of filiform macronuclei or associated singly or in groups with each bead of moniliform macronuclei. During cell division, filiform and moniliform macronuclei become compact and nucleoli dedif- ferentiate. Macronuclear division is accomplished primarily by extramacronuclear microtubules (Diener et al., 1983; Jenkins, 1973). These extrama- cronuclear microtubules may be intimately associ- ated with the cortex since De Terra (1983) has demonstrated cortical control over the direction of macronuclear elongation in Stentor . Since the majority of ciliates use intramacronuclear microtu- bules in macronuclear karyokinesis , Orias (1991a) has argued that extramacronuclear microtubules represent an independent evolution of the capacity to divide the macronucleus (see Chapter 4 ). Relatively little research has been done on the molecular biology of heterotrich nuclei. The macro- nuclear DNA molecules or “chromosomes” are typically long, some being up to 20 µm (Hufschmid, 1983; Pelvat & De Haller, 1976). Thus, very little frag- mentation of micronuclear chromosomes appears to occur, in contrast to other classes (Riley & Katz, 2001; Steinbrück, 1990). The heterotrich Blepharisma at least shows a deviation from the use of the universal stop codons – UAA, UGA, and UAG. Of the three, it uses at least UAA, like the spirotrich Euplotes (Liang & Heckmann, 1993) (see Chapter 7 ). As noted above (see Life History and Ecology ), conjugation may be initially stimulated by starvation conditions or changes in temperature. These condi- tions stimulate transcription of a gamone gene (Sugiura, Kawahara, Iio, & Harumoto, 2005), which is followed by excretion of diffusible mating type substances, called gamones (Miyake, 1996) or mating pheromones (Luporini & Miceli, 1986). All species of Blepharisma excrete two gamones: blepharismone , a tryptophan derivative resembling serotonin (Kubota, Tokoroyama,Tsukuda, Koyama, & Miyake, 1973; Miyake & Bleyman, 1976) com- mon to all species; and blepharmone , a 20-kDa glycoprotein that is species specific (Braun & Miyake, 1975). Typically, the diffusion of these substances is sufficient to stimulate conjugation in receptive individuals of fresh-water species of Blepharisma but is apparently not sufficient in marine species (Ricci & Esposito, 1981). There is considerable debate about the precise mechanisms that stimulate conjugation in hetero- trichs at the cellular and molecular levels. Miyake (1996) favors his gamone-receptor hypothesis while Luporini and Miceli (1986) reinterpret the results from Blepharisma in the context of their self-recogni- tion hypothesis . Whichever interpretation is true, the heterotrichs have not evolved a stable mating type system, as stable lines are quite rare (Demar-Gervais, 1971; Miyake & Harumoto, 1990). Luporini and Miceli (1986) argued that Blepharisma (and therefore heterotrichs in general?) has not yet evolved a mating type system and that it is only the formation of blep- harmone-blepharismone complexes that stimulate conjugation . Once conjugation is stimulated and micronuclear meiosis has occurred, a variable number of micro- nuclei enter the third or pregametic division: one in Spirostomum and Blepharisma americanum , but two or three in Fabrea and Blepharisma japonicum (Raikov, 1972). Ultimately, the products of only one of these micronuclei form the stationary and migratory gametic nuclei, which fuse to form the synkaryon . Thus, the exconjugants are genetically identical to each other although different from their parents. The synkaryon typically divides three times to produce eight products, several of which develop as macronuclear anlagen that may fuse to form the macronucleus, following separation of the conjugants (Raikov, 1972). 6.7 Other Features Given the widespread distribution of ciliates, and heterotrichs in particular, and their large cell size and ease of cultivation, several laboratories have developed low-cost bioassays or microbiotests using Spirostomum species. Spirostomum turns out to be quite a sensitive indicator species, especially to some heavy metal contaminants (Madoni, 2000; Nalecz-Jawecki, 2004; Nalecz-Jawecki & Sawicki, 2002; Twagilimana, Bohatier, Grolière, Bonnemoy, & Sargos, 1998). 6.7 Other Features 139
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