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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