Zoomorphology (2012) 131:127–148
DOI 10.1007/s00435-012-0149-0
ORIGINAL PAPER
Form and function of the feeding apparatus in Eutardigrada
(Tardigrada)
Roberto Guidetti • Tiziana Altiero • Trevor Marchioro • Luca Sarzi Amadè
Alexandra M. Avdonina • Roberto Bertolani • Lorena Rebecchi
Received: 8 November 2011 / Revised: 23 January 2012 / Accepted: 31 January 2012 / Published online: 6 March 2012
Ó Springer-Verlag 2012
Abstract Tardigrade feeding apparatus is a complex
structure with considerable taxonomic significance that can
be schematically divided into four parts: buccal ring, buccal
tube, stylet system, and pharynx. We analyzed the fine
morphology and the tridimensional organization of the
tardigrade buccal–pharyngeal apparatus in order to clarify
the relationships between form and function and to identify
new characters for systematic and phylogenetic studies. We
conducted a comparative analysis of the cuticular structures
of the buccal–pharyngeal apparatuses of twelve eutardi-
grade species, integrating data obtained by SEM and LM
observations. Morphological diversity was observed and
new cuticular structures such as the stylet coat of the stylet
system were identified. The synthesis of the buccal–pha-
ryngeal apparatus during molting was also analyzed
obtaining a clear developmental sequence of its resynthesis.
These findings lead us to redefine the previous interpreta-
tions of the functioning mechanisms of the buccal–pha-
ryngeal apparatus and provide a more specific relationship
Communicated by A. Schmidt-Rhaesa.
R. Guidetti
T. Marchioro
L. Sarzi Amadè

R. Bertolani (&)
L. Rebecchi
Department of Biology, University of Modena and Reggio
Emilia, Via Campi 213/D, 41125 Modena, Italy
e-mail: roberto.bertolani@unimore.it
T. Altiero
Department of Education and Human Sciences,
University of Modena and Reggio Emilia, Via Allegri 9,
42121 Reggio Emilia, Italy
A. M. Avdonina
The Russian Presidential Academy of National Economy
and Public Administration, Vladimir Branch,
Gorkogo St. 59A, 600017 Vladimir, Russia
•
between tardigrade diet and the anatomy of their feeding
apparatuses. In addition, the detection by energy-dispersive
X-ray spectroscopy of calcium in the stylets, buccal tube,
and placoids of eutardigrade species (i.e., Milnesium tar-
digradum, Paramacrobiotus richtersi) indicates that CaCO3
incrustations are not an exclusive feature of heterotardi-
grades and lead to suppose that this trait was present in the
ancestors of both classes.
Keywords Buccal–pharyngeal apparatus
Calcium
carbonate
Evolution
Foregut
Molting
X-ray
spectroscopy
Introduction
Tardigrades, or water bears, are microscopic metazoans
(100–1,000 lm in length), whose terrestrial species can be
desiccation-tolerant (Rebecchi et al. 2007; Guidetti et al.
2011). Tardigrades are divided into Eutardigrada and Het-
erotardigrada. Within eutardigrades, there are two mono-
phyletic taxa, Apochela and Parachela (Sands et al. 2008;
Guil and Giribet 2011), whereas the relationships within
heterotardigrades (Arthrotardigrada and Echiniscoidea) are
still debated (Jørgensen et al. 2010). Tardigrada have been
included in the clade Ecdysozoa together with nematodes
and other molting animals (Aguinaldo et al. 1997). A recent
phylogenomics analysis places tardigrades within Panar-
thropoda suggesting a sister group relationship between
Arthropoda and Onychophora (Campbell et al. 2011).
Tardigrades colonize a wide range of environments (sea,
freshwater, soil) in which they represent an important
component of the meiofaunal communities. They can be
predators, prey or primary consumers in food webs. More
specifically, some species prey on other micrometazoans,
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128 Zoomorphology (2012) 131:127–148

mainly nematodes and rotifers, but also on other tardi-
grades, and some species are herbivorous, feeding on plant
material such as algae and mosses, while others feed on
bacteria and detritus (for summary see Nelson et al. 2010).
Despite the wide range of food consumption, tardigrades
do not display a great variation in the organization of the
digestive system which consists of a foregut, a midgut, and
a hindgut. The foregut mainly consists of a sucking buccal–
pharyngeal apparatus and an esophagus internally lined by
cuticle. The midgut, the largest section of the digestive
tract, is the site of digestion. The hindgut has a rectum with
a cuticular lining opening in a true cloaca in eutardigrades
or in an anus in heterotardigrades. The buccal–pharyngeal
apparatus basically consists of cuticular structures such as a
buccal ring, a buccal tube (totally or partially rigid) with
apophyses for muscle attachments, stylets and related
supports, a muscular sucking pharynx (containing bars or
placoids), and two glands responsible, together with the
pharynx and esophagus, for rebuilding the cuticular struc-
tures during each molt (for summary see Nelson et al.
2010). In this paper, we report a comparative analysis of
the morphology of the buccal–pharyngeal apparatuses of
eutardigrades belonging to both Apochela and Parachela,
integrating data obtained by scanning electron microscopy
and light microscopy observations. For Parachela, we
consider species belonging to Macrobiotoidea (Sands et al.
2008) which includes 40% of parachelan species. In addi-
tion, we analyze the synthesis of the buccal–pharyngeal
apparatus during molting and the presence of calcium
in the buccal–pharyngeal apparatus in two species repre-
sentative of both eutardigrade orders. The tardigrade
buccal–pharyngeal apparatus is a complex structure
with considerable taxonomic significance among taxa
belonging to several taxonomic levels, particularly in the
eutardigrades (Guidetti and Bertolani 2001a, 2005; Nichols
et al. 2006; Pilato and Binda 2010). Since one of the main
problems in eutardigrade taxonomy is related to the low
number of useful characters, we provide a careful analysis
of the cuticular structures of the buccal–pharyngeal appa-
ratuses in order to reveal new useful characters for taxo-
nomic and phylogenetic studies. In addition, fine analyses
of tardigrade feeding structures were carried out to clarify
the relationship between form and function of these
structures for a better understanding of their evolution.
Materials and methods
Morphological analysis of the buccal–pharyngeal
apparatus
The anatomy of the buccal–pharyngeal apparatuses of 12
species belonging to 8 genera of Eutardigrada represen-
tative of both orders (Table 1) was examined by light
microscopy (LM) and scanning electron microscopy
(SEM).
The buccal–pharyngeal apparatuses of in vivo speci-
mens, or those mounted in polyvinyl lactophenol or in
Faure-Berlese fluids, were observed with a Leica Leitz DM
RB microscope at Phase Contrast (PhC) or Differential
Interference Contrast (DIC). To understand the relative size
of buccal–pharyngeal structures, the length and diameter of
the buccal tube, the level of the stylet support insertion on
the buccal tube and the length of the ventral lamina, all
related to the length of the buccal tube (Pilato 1981) were
considered (Table 2). In addition, the thickness of placoid
margins, the size of the buccal crown (see below), and the
stylet furca were also considered (Table 2).

Table 1 Analyzed species, the substrate from which they have been extracted and sampling sites
Taxon Species Substrate Sampling site

Apochela
Milnesiidae Milnesium cf. tardigradum Moss, lichen on tree Modena (Italy), Madagascar
Parachela
Macrobiotidae Paramacrobiotus richtersi (Murray 1911) Leaf litter, moss on tree Modena (Italy)
Macrobiotus sandrae Bertolani and Rebecchi 1993 Moss on tree Modena (Italy)
Macrobiotus joannae Pilato and Binda 1983 Leaf litter Modena (Italy)
Macrobiotus macrocalix Bertolani and Rebecchi 1993 Moss on rock Modena (Italy)
Macrobiotus islandicus Richters 1904 Moss on rock Lofoten Islands (Norway)
Minibiotus gr. intermedius Moss on tree Modena (Italy)
Minibiotus furcatus (Ehrenberg 1859) Moss on tree Modena (Italy)
Adorybiotus granulatus (Richters 1903) Moss on rock Lofoten Islands (Norway)
Richtersius coronifer (Richters 1903) Moss on rock Modena (Italy)
Murrayidae Murrayon pullari (Murray 1907) Moss in water Trento (Italy)
Dactylobiotus pathenogeneticus Bertolani 1982 Moss in water, algae Trento (Italy), Modena (Italy)

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Table 2 Diet, mouth position, size of buccal diameter and ventral thickness of placoid margins, buccal crown, and stylet furca, and
lamina relative to the buccal tube length, position of the stylet support amplitude of the curvature of the stylet of the considered species
insertion on the buccal tube relative to the buccal tube length, relative
Species Diet Mouth Buccal tube Ventral Stylet support Placoid Buccal Stylet Stylet
position diameter (%) lamina (%) insertion (%) margins crown furca curvature

Adorybiotus Vegetarian Ventral- 7–8 30–40 62–65 Large Large Large Small
granulatus anterior
Richterius coronifer Vegetarian Ventral- 7–8 30–35 62–65 Large Large Large Small
anterior
Minibiotus gr. Vegetarian Ventral- 7–8 13–18 49–54 Large Small Small Small
intermedius anterior
Minibiotus furcatus Vegetarian Ventral- 10–12 63–68 68–73 Large Small Small Small
anterior
Murrayon pullari Vegetarian Ventral- 10–12 46–49 72–77 Medium Medium Large Small
anterior
Dactylobiotus Vegetarian Ventral- 10–12 46–49 72–77 Small Medium Large Small
parthenogeneticus anterior
Macrobiotus spp. ? Anterior- 15–19 60–65 78–83 Small- Medium Small Medium
ventral medium
Paramacrobiotus Carnivorous Anterior- 23–25 68–73 78–83 Small Medium Small Wide
richtersi ventral
Milnesium cf. Carnivorous Anterior 40–50 Absent 58–63 Absent Absent Very Absent
tardigradum small

The buccal–pharyngeal apparatuses were prepared for
SEM observations with the sodium hypochlorite (NaClO)
extraction method suggested by Eibye-Jacobsen (2001a) or
were simply collected after their natural discharging during
the molting process. In the hypochlorite extraction method,
the animal body was disrupted with needles within a drop
of diluted NaClO solution. After the tissues around the
buccal apparatus have been destroyed, the apparatus was
collected with a glass pipette and transferred on a cover-
glass. Naturally discharged buccal tube was also trans-
ferred on a coverglass. Each buccal tube has been washed
in distilled water and left to dry on the coverglass which
was singularly mounted on a stub and sputter-coated with
gold–palladium. For SEM observations, a SEM XL 40 (Fei
Company-Oxford Instruments) available at the ‘Centro
Interdipartimentale Grandi Strumenti’ of the University of
Modena and Reggio Emilia was used.
Analysis of the cuticular structures of the buccal–
pharyngeal apparatus during molting
The pattern of resynthesis of the cuticular structures of the
buccal–pharyngeal apparatus during molting was analyzed
in Milnesium cf. tardigradum and Paramacrobiotus rich-
tersi as representatives of both eutardigrade orders. After
extraction from their substrate, ten animals of each species
were kept individually in small caps with natural mineral
water at 14°C until the discharge of the cuticular structures
of the buccal–pharyngeal apparatus. Subsequently, they
have been observed in vivo three times per day and pho-
tographed. Moreover, buccal–pharyngeal apparatuses have
been extracted from animals close to the end of the molting
process and prepared for SEM observations using the
hypochlorite method mentioned above.
Detection of calcium in the buccal–pharyngeal
apparatus
The presence of calcium in the buccal–pharyngeal
apparatuses of M. cf. tardigradum and P. richtersi was
identified by microanalyses. For each species, five buccal–
pharyngeal apparatuses extracted from animals with the
hypochlorite method were analyzed. For each apparatus, a
map of the elements was obtained together with calcium
presence in eight spots of the buccal–pharyngeal apparatus,
namely: the buccal ring (1; when present), the anterior
portion of the buccal tube (2; just after the mouth), the
middle portion of the buccal tube (3; at the level of the
stylet supports), the caudal end of the buccal tube (4),
the ventral lamina (5; when present or visible), the middle
portion of the piercing stylet (6), the condyle of the stylet
furca (7), and the first macroplacoid in the pharynx
(8; when present) (Table 3). As control (‘‘blank’’), an area
of the stub very close to the buccal tube was considered.
The presence of calcium in the structures of the buccal–
pharyngeal apparatus was detected by X-ray mapping and
X-ray area-analysis methods performed using energy-
dispersive X-ray spectroscopy (EDX) utilizing the X-EDS

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Table 3 Presence of calcium detected by X-ray spectroscopy (area-analysis method) in the buccal–pharyngeal apparatus of Paramacrobiotus
richtersi and Milnesium cf. tardigradum
Site of analysis (as in Paramacrobiotus richtersi Milnesium cf. tardigradum
Figures)
% of Relative concentration % of
presence (%) frequency

Control 0 0 0 0
Buccal ring 1 33 5 0
Buccal tube: anterior 2 100 13 0
Buccal tube: middle 3 100 16 0
Buccal tube: terminal 4 100 8 0
Ventral lamina 5 100 20 0
Piercing stylet 6 100 – 100
Stylet furca 7 100 15 0
Pharynx cuticular 8 100 6 0
lining

% of frequency = % of specimens in which calcium was detected; relative concentration = % of calcium concentration relative to the calcium
concentration recorded in the piercing stylet

Oxford INCA-350 system. The system was attached to an
environmental scanning electron microscope (ESEM)
Quanta-200 (Fei Company-Oxford Instruments) available
at the ‘Centro Interdipartimentale Grandi Strumenti’ of the
University of Modena and Reggio Emilia. When calcium
was detected, the EDX spectrum showed the highest peaks
of X-ray intensity at 3.690 keV (Ka) and 4.012 keV (Kb).
Results
Anatomy of the buccal–pharyngeal apparatus
We schematically divided the cuticular structures of the
buccal–pharyngeal apparatus (Fig. 1a) into four parts:
buccal ring, buccal tube, stylet system, and pharynx. They
are followed by an esophagus which is still part of the
foregut.
Buccal ring and mouth
The mouth (Fig. 2) is cuticular and is formed by the buccal
ring and by the rostral part of the buccal tube, down to the
openings of the stylet sheaths. The mouth opening is gen-
erally circular, but in Adorybiotus granulatus and Richter-
sius coronifer it is dorso-ventrally compressed and delimited
by two transverse laminar expansions (cuticular ‘‘lips’’)
(Figs. 3b, c, 4d). The buccal ring (or mouth ring) is con-
nected with the buccal tube through a thin cuticular flexible
flange (or ridge) (Fig. 1a), which allows its telescopic
movement. Anteriorly, the buccal ring bears peribuccal
structures (e.g., lamellae, papulae, velum–see below; Fig. 2)
and, only in parachelan species, the anterior band of the
buccal armature formed by an internal band of small protu-
berances (teeth or mucrones) present at the base of peri-
buccal structures (Figs. 1a, 4b–d, 7d, 9f). The buccal ring of
the apochelan Milnesium cf. tardigradum is mounted by
six large lamellae each one inserted on a semilunar structure
which has external longitudinal striations (Figs. 2a, 5b, 6b).
Instead, in the parachelan Paramacrobiotus richtersi,
Macrobiotus sandrae, Macrobiotus joannae, Macrobiotus
macrocalix, Macrobiotus islandicus, Murrayon pullari, and
Dactylobiotus parthenogeneticus, the buccal ring is mounted
by ten lamellae (Figs. 2b, 7c). The lamellae of M. pullari are
fused (Fig. 11d), while those of P. richtersi seem connected
to each other by a fine string of cuticle (Fig. 7c). In Mini-
biotus gr. intermedius and in Minibiotus furcatus, the buccal
ring is mounted by ten papulae (Fig. 2c). The buccal ring of
R. coronifer and A. granulatus bears a wide lamina (velum,
probably fused lamellae) with the anterior band of the buccal
armature (Fig. 2d). A line of pits can be seen on the external
surface of the buccal ring of M. cf. tardigradum and
P. richtersi (Figs. 5b, 7c).
Buccal tube
The buccal tube (or mouth tube) is a cylindrical and rigid
structure whose diameter varies according to the species
(Table 2). The buccal tube is straight in the apochelan
M. cf. tardigradum, while in parachelan species, it shows
generally an anterior-ventral bend (Figs. 7, 8, 9, 11,
12, 13). In A. granulatus and M. gr. intermedius, the pos-
terior portion of the buccal tube is also bent and two
small ventro-lateral crests are present on its wall in

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a b c surrounded by cuticular crests and laminae. For this system
Fig. 1 Schematic drawings of (a) an eutardigrade buccal–pharyngeal
apparatus and (b, c) stylet system of Milnesium, formed by stylet coat,
piercing stylet, and stylet support. a Stylet coat and buccal crown are
drawn in transparency to show the piercing stylet and the buccal
armature; b stylet system when the stylet protractor muscles are
relaxed; c stylet system when the stylet protractor muscles are
contracted (arrow heads folds of the stylet coat). aba anterior band of
the buccal armature; bc buccal crown; bl buccal lamellae; br buccal
ring; bt buccal tube; cf condyle of the furca; cp caudal process; fc
flange of cuticle; fi cuticular filament developing from furca condyle;
es esophagus; ma macroplacoids; mi microplacoids; pa pharyngeal
apophyses; pb pharyngeal bar; pba posterior band of the buccal
armature; ph pharynx; ps piercing stylet; rt rod-shaped thickening;
sc stylet coat; sf stylet furca; ss stylet sheaths; su stylet support;
tc transversal crests; vl ventral lamina
correspondence to this bend (Figs. 3a, 13a). In Milnesium,
the external surface of the buccal tube is wrinkled (Fig. 5b,
c), reflecting its internal trabecular fenestrated structure
(Fig. 6d, e) (for its ultrastructure see Dewel and Wallis
1973). In most specimens of M. cf. tardigradum, the buccal
tube has evident oval dips, one dorsal and one ventral,
close to the buccal ring (Fig. 5b).
The anterior portion of the buccal tube forms internally the
posterior part of the mouth cavity, which often shows the
posterior band of the buccal armature in the shape of a line of
large teeth in P. richtersi and a band of small teeth in
D. parthenogeneticus, M. pullari, A. granulatus, R. coronifer
(Figs. 4d, 2d), and in Macrobiotus species (Figs. 8f, 9f). This
band is followed by the dorsal and ventral transverse crests of
the buccal armature (Figs. 8f, 9c) also called ridges or in-
frabuccal baffles, and by the two holes of the stylet sheaths for
the passage of the piercing stylets.
Externally, the wall of the anterior portion of the buccal
tube, with the exception of Milnesium, is ventro-laterally
131
of crests, we propose the name ‘‘buccal crown’’ (Fig. 1a).
The buccal crown is very wide in R. coronifer (Fig. 4b, c)
and A. granulatus (Fig. 3b, d). In the last species posteri-
orly, it has two accessory cuticular expansions, long and
rod-shaped, located in correspondence to the stylet sheaths
(Fig. 3d). The buccal crown is wide in P. richtersi
(Fig. 7d), Macrobiotus species (Figs. 8, 9, 10), M. pullari
(Fig. 11b, c), and D. parthenogeneticus (Fig. 12c–e), while
it is small in Minibiotus species (Fig. 13). The buccal
crown is the structure for the attachment of the anterior
extremities of the protractor muscles of the stylets (paper in
preparation).
The most distal portion of the buccal tube ends within
the pharynx. The end of the buccal tube of Milnesium is
characterized by three triangular expansions located at
120° from each other, one ventrally and two dorso-laterally
(Fig. 5c). Between these expansions, three flexible ‘‘lami-
nar cuticular flaps’’ are present, each bearing a radial
laminar fold on its external side (Fig. 6g). These flaps, also
identified by TEM by Dewel and Clark (1973), work as
valves to close the buccal tube preventing any backflow of
food from the pharynx. In parachelan species, the end of
the buccal tube has an enlarged margin. In P. richtersi, the
posterior margin of the buccal tube has thickenings in the
shape of flat wings in correspondence to the pharyngeal
apophyses (see below), while it is arch-shaped in corre-
spondence to the placoid rows (Fig. 7a, b). In Macrobiotus
species and in Minibiotus furcatus, the distal margin of the
buccal tube is thick and forms small expansions in corre-
spondence to the pharyngeal apophyses, while it shows
indentations in correspondence to the placoid rows
(Figs. 8, 9, 10, 14). The end of the buccal tube of R. cor-
onifer and A. granulatus has two conical expansions that
are not symmetrical and are located only in correspondence
to the two dorso-lateral placoid rows (Figs. 3f, g, 4e, f).
In addition to the buccal crown, several other structures
can be associated with the external wall of the buccal tube
such as apophyses (e.g., dorsal and ventral crests or hooks)
for the insertion of the stylet protractor muscles, the ventral
lamina (strengthening bar), and the structures of the stylet
system (see below). The antero-dorsal portion of the buccal
tube of R. coronifer has a T-shaped huge apophysis formed
by a longitudinal crest bearing a transverse crest on its
anterior margin (Fig. 4a–c). A hole is present at the base of
the longitudinal crest (Fig. 4c), while four holes are present
on the anterior surface of the transverse crest (Fig. 4b). A
longitudinal crest-shaped apophysis with a large anterior
bulbous expansion is also present in the antero-dorsal
portion of the buccal tube of A. granulatus (Fig. 3a, b).
Four oval areas that we called ‘‘oval perforated areas’’
contain many small holes (about 0.1 lm). These are sym-
metrically oriented on each side of the stylet sheaths, one
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132
Fig. 2 Mouth opening (SEM).
a b
a Milnesium cf. tardigradum,
peribuccal lamellae (asterisk)
and oral sensory papillae
(arrow); b Paramacrobiotus
richtersi, peribuccal lamellae
(asterisk); c Minibiotus
furcatus, buccal papulae
(asterisk); d Richtersius
coronifer, buccal velum
(asterisk). aba anterior band of
the buccal armature; pba
posterior band of the buccal
armature
c d
dorsal and one ventral (Figs. 5d, e, 8e, 9e). The oval per-
forated areas are particularly wide in Milnesium (Fig. 5d,
e), while in parachelan species, they can be totally or
partially covered by the crests of the buccal crown
(Figs. 8e, 9e).
The ventral lamina, present in all parachelan species here
examined, is a crest-shaped structure with enlarged distal
margin whose relative length varies according to the species
(Table 2). Its anterior part is enlarged and has lateral
expansions that, according to the definition of Pilato and
Binda (2010), are called ‘‘caudal processes’’ (Figs. 8c, 9c,
10c, 13c). When the ventral lamina is long, it usually ends
with a bifurcation whose branches reach the stylet support
insertions (Figs. 7a, b, 9a, 10b, 12b). The proximal margin of
the ventral lamina is fused with the buccal tube, with the
exception of R. coronifer and M. gr. intermedius (Figs. 4c,
13c). The anterior part of the ventral lamina of R. coronifer,
A. granulatus, M. pullari, and D. parthenogeneticus exhibits
a bulbous expansion (Figs. 3b, 4c, 11c, 12d, e). This
expansion shows a deep invagination in M. pullari, D. par-
thenogeneticus, and R. coronifer. At the level of the con-
nection between the ventral lamina and the buccal crown, a
depression is present in P. richtersi, M. joannae, and M. is-
landicus (Figs. 7b, 9c, 10c).
Stylet system
On both sides of the buccal tube, there is the ‘‘stylet sys-
tem’’ made up of stylets and stylet supports. Differently
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Zoomorphology (2012) 131:127–148
from existing literature, we observed that each stylet is
composed of two parts, namely the ‘‘stylet coat’’ and the
‘‘piercing stylet’’ (Fig. 1).
The stylet coat is characterized by an anterior cylindrical
portion corresponding to the traditional stylet sheath and a
posterior portion corresponding to the traditional stylet
furca connected by a very thin layer creating a continuous
‘‘stylet coat’’ (Figs. 1b, c, 6c, 14b–d). Its presence is
demonstrated by the absence of a clear demarcation
between the stylet sheath and the stylet furca (Figs. 3d, 4c,
7d, 8e, 9e, 10c, 12c, 14b–d) and the formation of folds on
the stylet surface during the piercing stylet movements
(Fig. 6c). The stylet sheath is fused with the buccal tube
and externally reinforced by a rod-shaped cuticular thick-
ening (Fig. 1). The stylet furca is characterized by two
condyles with a wrinkled surface for the attachments of the
stylet protractor and retractor muscles. The piercing stylet
is a needle-like structure of calcium carbonate (see below)
enveloped by the stylet coat (Fig. 1). In M. cf. tardigrad-
um, the piercing stylet is thin and short (Fig. 6c) and never
reaches the stylet furca. In parachelan species, the piercing
stylet is long with a sword-shaped anterior extremity
(Fig. 12e), showing a jagged margin (Fig. 8d), and an
enlarged posterior extremity which is jammed within the
stylet furca (Fig. 14a).
The anatomy of the stylet differs among the analyzed
species. In M. cf. tardigradum, the stylets are straight and
run almost parallel to the buccal tube and have a thick
stylet coat showing an expansion called the stylet
Zoomorphology (2012) 131:127–148 133

Fig. 3 Adorybiotus granulatus,

a
buccal–pharyngeal apparatus
(SEM). a apparatus in toto,
lateral view; the arrow indicates
the ventro-lateral crest;
b anterior portion of the buccal–
pharyngeal apparatus with large
buccal crown and cuticular lip
(arrow); the buccal ring is not
present; c cuticular lips (cl) and
oval perforated area (arrow);
d ventral lamina; e stylet c
b
support, cuticular fibers (arrow
head) and stylet furcae (arrow
proximal apophyses);
f asymmetrical end of the buccal
tube and cuticular structures of
the pharynx; g external surface
of the cuticle (arrow) covering
the pharynx. bc buccal crown;
vl ventral lamina

d e

f g

‘‘elbow’’ (according to Eibye-Jacobsen 2001a) (Figs. 5f,
g, 6d). A hole, never described before, is present at the
posterior base of the stylet elbow (Figs. 5f, g, 6c) and
could represent the site through which salivary secretions
reach the prey (as hypothesized by Dewel and Wallis
1973) or the site through which the piercing stylet is
introduced within the stylet coat. If the mounting medium
dissolves the piercing stylet, a canal within the stylet coat
is clearly visible (Fig. 6c). In Milnesium, the condyles of
the small triangular stylet furcae terminate with bent tips
from which fine cuticular filaments develop caudally
(Figs. 5f, 6f).
In parachelan species here examined, the piercing stylet
runs within the stylet coat positioned in a furrow delimited
by the ventral lamina and the ventral wall of the buccal
tube (Figs. 7b, d, 9a, 10b, c, 12b). The stylet sheaths are
located ventrally to the buccal tube, while the stylet furcae
are located laterally to the buccal tube (Figs. 3, 4, 7, 8, 9,
10, 11, 12, 13). In P. richtersi, Minibiotus, and Macro-
biotus species, the stylets are bent and slightly dorso-ven-
trally compressed with a small crest just before the stylet
furca (Figs. 7a, 8b). In P. richtersi and Macrobiotus spe-
cies, the stylet furca has two short, large, and flat condyles
(Figs. 7, 8, 9, 10, 11), in M. islandicus, they show proximal
apophyses (Fig. 10a, b), while in Minibiotus species, they
are thumb-shaped (Fig. 13). In A. granulatus and R. cor-
onifer, the stylets are conical, almost straight, and slightly
laterally compressed (Figs. 3, 4). Their stylet furca is large
and stumpy, with two long and wide condyles forming
a very wide, almost straight arc (Figs. 3e, 4f). In

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134 Zoomorphology (2012) 131:127–148

Fig. 4 Richtersius coronifer, a

buccal–pharyngeal apparatus
(SEM). a apparatus in toto,
lateral view; b mouth opening
and buccal crown (arrow holes
in the T-shaped dorsal crest);
c buccal crown, dorsal crest
(white arrow) with basal hole
(black arrow), ventral lamina,
and stylet sheath; d cuticular
lips (cl) and oval perforated area
b c
(arrow); e asymmetrical end of
the buccal tube and external
surface of the cuticle (arrow)
covering the pharynx; f stylet
furcae. pba posterior band of the
buccal armature; bc buccal
crown; bt buccal tube; ma
macroplacoids; pa pharyngeal
apophyses; ss stylet sheaths;
vl ventral lamina d
e

A. granulatus, each condyle has an irregular triangular
shape and includes a proximal apophysis (Fig. 3e). In
R. coronifer, the condyles of each stylet furca have dif-
ferent shapes: the ventral condyle is round and compressed,
while the dorsal one is cylindrical and bent (Fig. 4f). In M.
pullari and D. parthenogeneticus, the stylets are slightly
bent, conical and without apparent lateral compression
(Figs. 11, 12). Their stylet furca is large, with two long and
slender condyles (Figs. 11e, 12a, b) that have proximal
apophyses in D. parthenogeneticus (Fig. 12a, b).
In all examined species, the stylet support is a flexible
cuticular structure that connects the buccal tube with the
stylet furca (Figs. 3, 4, 5–13). The level of the point of
stylet support insertion on the buccal tube can be species-
specific (Table 2). The stylet support is fused to the stylet
furca in correspondence of the arc between its two con-
dyles. Cuticular fibers also seem to be involved in
anchoring the stylet support to the stylet furca in M. cf.
tardigradum and A. granulatus (Fig. 3e), as already
detected in Dactylobiotus octavi Guidetti, Altiero and
Hansen, 2006 (Guidetti et al. 2006) and Halobiotus crispae
Kristensen, 1982 (Eibye-Jacobsen 2001a). In M. cf. tar-
digradum, the stylet support is short and triangular
(Fig. 5a, f, g), it can move backward up to 90° becoming
aligned with the buccal tube (Fig. 5f). In contrast, in all
parachelan species, the stylet support can move backward
but never becoming aligned with the buccal tube. The
stylet support shows a longitudinal groove in P. richtersi
and Macrobiotus species (Figs. 7, 8, 9, 10); it is thin but
with a thicker rigid portion in its proximal part in

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Zoomorphology (2012) 131:127–148 135

Fig. 5 Milnesium cf.

a
tardigradum, buccal–
pharyngeal apparatus (SEM).
a apparatus in toto, and cuticle
of pharyngeal lumen (asterisk),
ventral view; b buccal ring with
striated lamellae and a line of
pits (arrow), and fenestrated
zone of buccal tube (asterisk);
c posterior portion of the buccal
tube, dorsal view; d stylet
sheaths and oval perforated area
(arrow); e, perforated area;
f stylet support, posterior
portion of the stylet coat in
lateral view, elbow (asterisk), b c
and hole (arrow); g posterior
portion of the stylet coat in
frontal view, elbow (asterisk),
hole (arrow). br buccal ring; fi
cuticular filament developing
from furca condyle; ss stylet
sheaths; su stylet support

d e

f g

M. islandicus, R. coronifer and A. granulatus (Figs. 3e, 4a,
10b), and it is thick with a longitudinal groove and a rigid
proximal portion in M. pullari and D. parthenogeneticus
(Figs. 11e, 12b).
Pharyngeal structures and esophagus
The buccal tube ends within a myoepithelial pharynx
(Fig. 1a) with a tripartite lumen covered with a thin
cuticular lining that is connected to the buccal tube.
Within the pharynx, with the exception of Milnesium
(Figs. 5a, 6a), there are cuticular thickenings: three pha-
ryngeal apophyses that alternate with three rows of a
double series of symmetrical cuticular placoids. The
placoids can be separated into macroplacoids (2 or 3 per
line) and microplacoids (1 per line, when present, the
smallest and the most caudal) (Fig. 1a). Other cuticular
structures sometimes present in the pharynx are cuticular
rods pointing antero-laterally and aligned with placoids,
called ‘‘pharyngeal bars’’ (Fig. 7b) according to Eibye-
Jacobsen (2001b), and identified by LM also in A. gran-
ulatus, M. pullari, and D. parthenogeneticus. Macrobio-
tus islandicus, R. coronifer, A. granulatus, M. pullari,
and Minibiotus species have thick placoids (Figs. 3f, g,
4a, e, 10d, 11a, 13), while P. richtersi, M. macrocalix,
M. joannae, M. sandrae, and D. parthenogeneticus
have relatively thinner placoids (Figs. 7, 8, 9, 12b).
The pharyngeal apophyses are bilobed in P. richtersi,
Macrobiotus species, R. coronifer, A. granulatus, and
M. pullari. In addition, they have large margins in

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136 Zoomorphology (2012) 131:127–148

Fig. 6 Milnesium cf. a b

tardigradum, buccal–
pharyngeal apparatus (DIC).
a apparatus in toto of a live
specimen; b–g apparatuses of
fixed specimens. b pits (white
arrow) on buccal ring,
semilunar structures with
striations (black arrow); c stylet
system (enlargement of
g) showing fold in stylet coat
(black arrow) and hole in stylet
c
coat (white arrow); d,
e trabecular structures (arrows)
inside buccal tube wall: d lateral
view showing the stylet elbow
(asterisk), e dorsal view; f stylet
furca (lateral view); g apparatus
in toto showing laminar flaps
(arrow). fi cuticular filament
developing from furca condyle;
bt buccal tube; ph pharynx; ps
piercing stylet; sf stylet furca; su
stylet support
d e

f g

M. islandicus, R. coronifer, A. granulatus, M. pullari, and
Minibiotus species. The surface of the cuticle covering
the pharyngeal lumen is wrinkled in A. granulatus and
R. coronifer (Figs. 3g, 4e).
The cuticular lining of the pharynx continues within the
narrow cylindrical esophagus (Fig. 1a). At the transition
area between the pharynx and esophagus, there are folds
which work as a valve. These structures have been observed
in apochelan (Fig. 16g) and parachelan species (Dewel and
Clark 1973; Eibye-Jacobsen 1997; Avdonina et al. 2007).
Buccal–pharyngeal apparatus re-synthesis
In P. richtersi and M. cf. tardigradum, the two species
examined in regard to molting, the first visible clue of the
molting process is the discharging of the entire cuticular
lining of the foregut through the mouth opening. The fol-
lowing ‘‘simplex stage’’ is characterized by the absence of
any cuticular structures in the feeding apparatus (Figs. 15a,
16a), the mouth opening is closed by cuticular folds, and
the animal cannot feed. For both species, at 14°C, the
complete resynthesis of the cuticular structures of the
buccal–pharyngeal apparatus takes about 48–72 h.
During molting, the buccal tube and the stylet system
are produced by the salivary glands, and the posterior part
of the feeding apparatus is reconstructed by the pharynx
and the epithelia of the esophagus. The new structures are
resynthesized in a more posterior position with respect to
the place occupied in the body cavity of the non-molting
animals (Figs. 15, 16). Buccal–pharyngeal apparatus

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Zoomorphology (2012) 131:127–148 137

Fig. 7 Paramacrobiotus
richtersi, buccal–pharyngeal
apparatus (SEM). a in toto
apparatus naturally discharged
during molting, ventro-lateral
view; b ventral view of in toto
apparatus showing pharyngeal
bar (white arrow) and
invagination (black arrow)
between buccal crown and
ventral lamina; c buccal ring
with buccal lamellae, fine string
of cuticle (white arrow) and line
of pits (black arrow); d ventral
lamina and stylet sheaths. aba
anterior band of the buccal
armature; bc buccal crown; bl
buccal lamellae; br buccal ring;
ss stylet sheaths; vl ventral
lamina

resynthesis is the result of a series of continuous changes c). The posterior portion of the buccal tube is
that for the purpose of clarity we schematize in steps. reconstructed within the pharynx, therefore by the
pharyngeal cells and not by the salivary glands. The
1. After the discharging of the cuticular structures, the
formation of the buccal tube seems to proceed by the
two salivary glands move from a lateral to an anterior
aggregation of long, thin and rod-shaped longitudinal
position with respect to the pharynx and come into
portions. At this stage, all these structures suddenly
contact with each other (Figs. 15a, b, 16a, b) encom-
dissolve when the molting animals are mounted on
passing the muscles responsible of the stylet move-
slides with Faure-Berlese’s mounting medium.
ment. During molting, the pharynx is more deformable
3. In P. richtersi, the buccal armature is completely
than in the non-molting phase (Figs. 15d, 16g). In
formed (Fig. 15g) before the complete stiffening of
Milnesium, from the beginning of the resynthesis phase
the buccal tube. In both species, the outline of the
till its end, granules are visible where the buccal tube
buccal lamellae on the buccal ring is visible together
will be outlined and remain confined within the
with the ventral lamina (when present) even though
reconstructing buccal tube (Fig. 16b).
its anterior portion is not yet synthesized (Figs. 15e, f,
2. The anterior portion of the piercing stylet is produced
16c, d). At the posterior end of the buccal tube
together with the outlines of the buccal tube and the
(within the pharynx), structures ascribable to the
buccal ring that are initially flexible (Figs. 15b–d, 16b,

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138 Zoomorphology (2012) 131:127–148

Fig. 8 Macrobiotus sandrae,

a
buccal–pharyngeal apparatus
(SEM). a apparatus in toto,
dorsal view, without buccal
ring; b apparatus in toto, lateral
view; c ventral lamina showing
caudal process (arrow);
d piercing stylet with a jagged
margin (arrow); e stylet sheath
and oval perforated areas
(arrows); f posterior portion of
the buccal armature with
transverse crests (asterisks) and
oval perforated areas (arrows).
bc buccal crown; pba posterior
band of the buccal armature; ss
stylet sheaths; vl ventral lamina b

c d

e f

buccal tube valves are visible (Figs. 15i, 16c). The
piercing stylets are gradually formed, starting from
small quadrilateral crystals that elongate in an antero-
posterior direction and progressively move within the
stylet sheaths of the stylet coat that is forming
(Figs. 15d, e, 16c, d).
4. The stylet support outline is visible, showing a distal
bifurcation. The stylets are not yet complete: the rod-
shaped reinforcements of the stylet sheaths are formed,
while the posterior portions of the stylet coats (the
stylet furcae) are just outlined (Figs. 15e, f, 16d, e).
The piercing stylet is not completely formed, but its
anterior tip is completed. The buccal ring with the
buccal lamellae is complete. In P. richtersi, the
pharyngeal macroplacoids are outlined and the apoph-
yses are not yet visible (Fig. 15e).
5. The stylet furcae and the stylet supports develop
progressively. In P. richtersi, the macro- and micro-
placoids, and the pharyngeal apophyses develop
together with the stylet supports (Fig. 15f). The
piercing stylets are almost complete but with still
incomplete posterior extremities. The buccal tube is
not completely rigid (Figs. 15f, 16e). In P. richtersi,
the anterior portion of the ventral lamina is almost
completed.
6. When the buccal–pharyngeal apparatus is completely
formed, the process of sclerification of the cuticular
structures is almost finished, the salivary glands move

123
Zoomorphology (2012) 131:127–148 139

Fig. 9 Macrobiotus joannae

(a–c) and Macrobiotus
macrocalix (d–f) buccal–
pharyngeal apparatuses (SEM).
a apparatus in toto, ventral
view. b posterior portion of the
buccal tube, apophyses and
macroplacoids in the pharynx;
c ventral lamina with
invagination (arrow), stylet
sheaths, and transverse crest
(asterisk); d apparatus in toto,
lateral view; e stylet sheaths and
oval perforated area (arrow);
f buccal ring without buccal
lamellae. aba anterior band of
the buccal armature; bc buccal
crown; br buccal ring; bt buccal
tube; ma macroplacoids; pa
pharyngeal apophyses; pba
posterior band of the buccal
armature; ss stylet sheaths;
vl ventral lamina

laterally to the pharynx, and the apparatus is moved by Calcium detection in the buccal–pharyngeal apparatus
the associated muscles to the anterior final position
(Fig. 15g). Calcium has been detected by the method of X-ray area-
analysis spectroscopy in all analyzed structures of the
Concomitantly with the synthesis of the sclerified
buccal–pharyngeal apparatuses of P. richtersi (Table 3).
structures of the buccal–pharyngeal apparatus, a new body
The piercing stylets showed a very high concentration of
cuticle, including the esophagus and hindgut lining, is
calcium, from 5 to 16 times higher than that recorded in the
synthesized by the underlying epidermis or surrounding
other structures of the buccal–pharyngeal apparatus
epithelium and the new claws are produced by claw (pedal)
(Table 3). In Milnesium, the X-ray area-analysis method
glands in the legs. When cuticular production is complete
detected calcium only in the piercing stylets, and no traces
(new exoskeleton, including a new set of claws, and new
of calcium were detected in the other structures of the
internal sclerified structures), the animal performs the
buccal–pharyngeal apparatus (Table 3). The X-ray map-
ecdysis process abandoning the old cuticle (including the
ping spectroscopy method had a lower resolution than the
lining of the hindgut).
area-analysis method, producing a map of the presence of

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140 Zoomorphology (2012) 131:127–148

Fig. 10 Macrobiotus a
islandicus, buccal–pharyngeal
apparatus (SEM). a apparatus in
toto, dorsal view; b apparatus in
toto, ventral view (arrow
proximal apophyses); c stylet
sheaths and ventral lamina with
invagination (arrow);
d posterior portion of the buccal
tube, apophyses and
macroplacoids in the pharynx.
br buccal ring; bt buccal tube;
ma macroplacoids; pa
pharyngeal apophyses; ss stylet
sheaths; vl ventral lamina

c d

calcium only for piercing stylets in which calcium was in
very high concentration (Fig. 17).
Discussion
The comparison of the sclerified structures of the tardi-
grade feeding apparatuses of several eutardigrade taxa,
analyzed combining SEM and in vivo LM observations,
allowed us to define their fine morphology, obtain a better
understanding of their tridimensional organization, and
identify new structures. These new findings lead us to
redefine the previous interpretations of the functioning
mechanisms of this apparatus.
Some considerations can be offered about several parts
of the buccal–pharyngeal apparatus.
Mouth and buccal ring
The mouth, in particular its buccal ring, is articulated with
the following buccal tube, allowing telescopic and tilting
movements, enabling a more efficient sucking action with
the adhesion of the mouth to the substrate, facilitated by its
peribuccal structures. Solely in the apochelan Milnesium,
the lamellae work as a shutter. As pointed out by Kris-
tensen (1987) for heterotardigrades, the flexible joint areas
between mouth and buccal tube are also useful to avoid
animal cuticle breakages during the severe longitudinal
shrinking of the animal during the desiccation process. The
pits on external surface of the buccal ring are pores through
which the sensilla of the sub-oral sensory region (SOS)
cross the buccal ring cuticle entering the mouth cavity
(Dewel and Eibye-Jacobsen 2006). The teeth of the buccal

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Zoomorphology (2012) 131:127–148
Fig. 11 Murrayon pullari,
buccal–pharyngeal apparatus
(SEM). a apparatus in toto,
lateral view; b stylet sheaths
with rod-shaped cuticular
thickening (arrow); c buccal
crown; d buccal ring with fused
buccal lamellae (asterisk);
e stylet furca. bc buccal crown;
br buccal ring; ss stylet sheaths
armature could be used to hold food during its ingestion,
while the transverse crests could prevent mechanical
damage of the buccal sensory organ during food ingestion.
More clearly, the cuticular ‘‘lips’’ and the wide bands of
teeth present on the velum in A. granulatus and R. coro-
nifer can be thought as a spatula for grazing food from the
substrate.
Buccal tube
As regards the buccal tube, in parachelan species, the
buccal crown is the structure for the attachment of the
anterior extremities of the protractor muscles of the sty-
lets, which correspond to the muscles M1 and M6 of the
stylet system (Marcus 1929; Schmidt-Rhaesa and Kulessa
2007; Halberg et al. 2009). The buccal tube of Milnesium
apparently does not possess any specialized structures for
this function; therefore, in apochelan species, the anterior
attachment for the protractor muscles of the piercing
stylets remains to be identified. The oval dips of the tube
in Milnesium correspond to the ‘‘fenestrated zone’’ iden-
tified by Dewel and Eibye-Jacobsen (2006) in this genus,
but differently from these authors, we never found per-
forations. On the other hand, in all tardigrades, the
retractor muscles of the stylets are connected to the
condyles of the furca and to the surface of the pharynx
141
(Marcus 1929; Schmidt-Rhaesa and Kulessa 2007; Hal-
berg et al. 2009).
The four ‘‘oval perforated areas’’ of the buccal tube are
the regions through which the microvillus-like sensory
processes of the sensitive pharyngeal organs (or buccal
sensory organs; Dewel and Wallis 1973; Walz 1978, 1979;
Dewel and Eibye-Jacobsen 2006) cross the buccal tube and
reach the inner surface of the mouth at the base of the
transverse crests of the buccal armature (Fig. 8f), or at the
base of the cuticular lips (Figs. 3c, 4d).
Relationships between diet and the anatomy
of the feeding apparatus
Hallas and Yeates (1972) hypothesized a correlation
between the width of the buccal tube in various species and
their diet. Our data support this hypothesis and provide a
more specific relationship between diet and the anatomy of
the feeding apparatus. The buccal–pharyngeal apparatus of
the eutardigrade species that suck fungi and bacteria or cell
contents of moss and/or lichens (e.g., R. coronifer) gener-
ally have: (1) a narrow and long buccal tube, (2) stylet
supports inserted more anteriorly on the buccal tube, (3)
thick placoids with wide distal margins, (4) large pharyn-
geal apophyses, (5) large stylet furcae, (6) a large surface
of the buccal crown and/or large apophyses for the
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142 Zoomorphology (2012) 131:127–148

Fig. 12 Dactylobiotus
parthenogeneticus, buccal–
pharyngeal apparatus (SEM).
a apparatus in toto, dorsal view;
b ventral view of an apparatus
in toto, apophyses of the furcae
visible (arrow); c buccal crown
and stylet sheaths; d buccal
crown, stylet sheaths in lateral
view, and ventral lamina with
invagination (arrow); e piercing
stylets out of the buccal tube
and ventral lamina with
invagination (arrow). bc buccal
crown; ps piercing stylet; ss
stylet sheaths; vl ventral lamina

insertion of the stylet muscles, and (7) a ventro-anterior
mouth (Table 2). Instead, the buccal–pharyngeal apparatus
of carnivorous species [e.g., M. cf. tardigradum and
P. richtersi, among those here considered (see Table 2),
and Bertolanius weglarskae (Dastych 1972) (see Guidetti
and Bertolani 2001b)] is characterized by: (1) a wide and
relatively short buccal tube, (2) a large pharynx either
without placoids or with thin placoids, and (3) large buccal
lamellae on the wide mouth ring. In the parachelan species
here examined, a relationship between the size of the stylet
furcae and the size of the buccal crown can be seen: the
wider the stylet furcae the wider the buccal crown
(Table 2). This relationship, previously noted in Boreali-
bius (Parachela, Hypsibiidae) (see Pilato et al. 2006), is due
to the fact that the same protractor muscles (M1 and M6)
are attached to both structures. Wide furcae and wide
buccal crown allow the presence of large muscles which
are probably related to a greater penetration power of the
piercing stylets. Piercing stylets are used to remove the
food from substrates (e.g., bacteria, fungi), to penetrate
the plant cells wall, or to capture the preys (before to
swallow them) or/and broke the animal cuticles to suck the
internal fluids. An increase in food consumption is also
obtained by increasing the sucking power of the pharynx.
As observed by Eibye-Jacobsen (2001b), this increase is
obtained by increasing the number of perradial muscle cells
of the pharynx which is related to an increase in the surface
at which the cells are attached. Macroplacoids that are very
thick (e.g., R. coronifer, A. granulatus, Minibiotus spp.)
have wide distal margins and therefore a wider surface for

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Zoomorphology (2012) 131:127–148 143

Fig. 13 Minibiotus gr.

intermedius (a–c) and
Minibiotus furcatus (d, e),
buccal–pharyngeal apparatuses
(SEM). a dorsal view of in toto
apparatus showing the ventro-
lateral crests (arrow);
b apparatus in toto, ventral
view; c ventral lamina showing
caudal process (arrow);
d apparatus in toto, lateral view;
e apparatus in toto, dorsal view.
vl ventral lamina

the attachment of the perradial muscle cells leading to an
increase in the sucking power of the pharynx. A reduced
size of the buccal diameter, associated with a higher
sucking power of the pharynx, leads to a further increase in
the suction pressure at the level of the mouth. In agreement
with this hypothesis, eutardigrades without placoids (e.g.,
Milnesium) or with reduced placoids (e.g., Itaquasconinae),
and large buccal tubes have developed a large pharynx to
increase its sucking power.
Morphology and function of the stylet system
According to this study, the morphology and function of
the stylet system appears in a new light. The stylet system
is normally neglected or overlooked because the piercing
stylets usually disappear in dead or fixed tardigrades. The
presence of a stylet coat, and in particular of its middle
portion connecting the stylet sheath to the stylet furca
(Fig. 1b), is a new result for tardigrades. In the traditional
view, the stylet sheath was considered completely sepa-
rated from the stylet furca. In previous analyses, the middle
portion of the stylet coat was overlooked probably due to
the fact that it can easily disappear due to its highly
reduced thickness and/or chemical composition. Never-
theless, a careful analysis and interpretation of data and
images reported by a few authors does provide evidence of
the presence of a continuous stylet coat in species beyond
those examined in this paper. For example, the SEM

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144 Zoomorphology (2012) 131:127–148

Fig. 14 Stylet coat. a buccal–

pharyngeal apparatus of
Paramacrobiotus richtersi in
vivo in which the piercing
stylets (asterisk) are visible
(DIC); b–d ventral view of
buccal–pharyngeal apparatuses
in mounted specimens in which
the piercing stylets are dissolved
and the empty stylet coats are
visible (arrows) (PhC);
b Paramacrobiotus richtersi;
c Richtersius coronifer;
d Macrobiotus macrocalix

pictures of the buccal–pharyngeal apparatuses of the het-
erotardigrade Echiniscoides sigismundi (Schulze 1865) and
the eutardigrade Halobiotus crispae Kristensen 1982 (in
Eibye-Jacobsen 2001a: Figs. 6, 9) show a structural con-
tinuity between the stylet sheath and the stylet furca that is
in agreement with our finding. The SEM images of the
buccal–pharyngeal apparatuses of the eutardigrades Dac-
tylobiotus grandipes (Schuster et al. 1978) (in Schuster
et al. 1980: Fig. 10) and Thulinius saltursus (Schuster et al.
1978) (in Schuster et al. 1980: Fig. 12; Dewel et al. 1993:
Fig. 25) show thin layers connecting the stylet furca to the
stylet sheaths. These connections were interpreted as a
‘‘stylet retainer’’ by Schuster et al. (1978), but they have to
be reinterpreted as traces of the empty stylet coats. The
very thin layer of cuticle surrounding the piercing stylets of
the eutardigrade Ramazzottius tribulosus Bertolani and
Rebecchi, 1988 identified by TEM by Avdonina et al.
(2007) represents the stylet coat. Finally, the pseudosimplex
stage of H. crispae shows a reduced piercing stylet and a
cuticular covering persisting in polyvinyl-lactophenol-
treated animals (Kristensen 1982). This covering should be
identified as the stylet coat. Therefore, when we refer to the
stylet, we consider the stylet coat, composed by the stylet
sheaths and the stylet furcae which are in structural conti-
nuity, and the piercing stylet positioned within it (Fig. 1).
The new delineation of the anatomy of the stylet system,
including the discovery of new structures such as the stylet
coat, leads us to redefine its functioning mechanism that

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Zoomorphology (2012) 131:127–148 145

Fig. 15 Paramacrobiotus
richtersi (DIC). Phases of
synthesis of the buccal–
pharyngeal apparatus during
molting process (live
specimens). a ‘‘Simplex stage’’
characterized by the absence of
sclerified structures; b anterior
portion of piercing stylets in
formation (white arrow),
outlines of buccal tube (arrow
head) and buccal ring;
c successive step of the previous
phase (b); d step in which
cuticular structures and pharynx
are highly deformable; e ventral
lamina and stylet support in
formation (arrow), and piercing
stylet almost completed; f stylet
furcae, stylet supports
bifurcated at their extremities
(arrow), placoids and apophyses
in the pharynx not yet complete;
g complete buccal–pharyngeal
apparatus in its final position;
h buccal armature in formation,
transverse crests are visible
(arrow); i posterior end of
buccal tube (within pharynx)
with structures ascribable to
buccal tube valves (arrow)
(enlargement of Fig. 16c).
asterisk salivary glands; black
arrow buccal ring; aba anterior
band of the buccal armature;
pba posterior band of the buccal
armature; ph pharynx

can be schematized as follows. When the protractor mus-
cles pull the stylet furcae, the piercing stylet is pushed out
of the stylet coat through the holes of the stylet sheath and
enters the mouth cavity (Fig. 1c). This movement produces
folds on the surface of the stylet coat at the level of its
middle portion (Figs. 1c, 6c). The stylet coat plays the role
of isolating the piercing stylet from the body cavity as it
operates outside the mouth cavity. In this way, the stylet
coat inhibits any external substances or organisms
(including pathogens) from penetrating within the body
cavity through the stylet sheath holes. We can also consider
the relationship between the shape of the stylets and the
length of the ventral lamina. We found that with longer
ventral lamina, the stylets are more bent (Table 2). The
long ventral lamina of P. richtersi and of the Macrobiotus
species works to guide the bent stylets during their
movements keeping them in the right position. In
R. coronifer and A. granulatus, in which the stylets are
straighter, the ventral lamina is shorter and looses its guide
function. In conjunction with the ventral lamina, also the
stylet sheath, the stylet support, and the protractor and
retractor muscles aid in keeping the piercing stylets in the
right position and directing their movements. Moreover,
the stylet support works as a fulcrum for stylet movement.
As pointed out by Marcus (1929) and Dewel and Wallis
(1973), the broader arc covered by the stylet supports in
apochelan compared to parachelan species increases the
range of the stylet movement compensating for the
unusually short stylets present in apochelans.
Buccal–pharyngeal apparatus during molting
Molting is a phenomenon occurring periodically through-
out the life of tardigrades. During molting of M. cf.

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146 Zoomorphology (2012) 131:127–148

Fig. 16 Milnesium cf. a b

tardigradum. Phases of
synthesis of buccal–pharyngeal
apparatus during molting
process (a–e, g: live specimens;
f fixed specimen). a ‘‘Simplex
stage’’ characterized by the
absence of sclerified structures;
b anterior portion of piercing
stylet in formation (white
arrow), outlines of buccal tube
with granules (arrow head);
c stylet support in formation
(white arrow), piercing stylet c d
and buccal ring almost
completed, buccal tube valves
visible (arrow head); d buccal–
pharyngeal apparatus almost
completed with visible stylet
coat (arrow head); e successive
step of previous phase (d);
f complete buccal–pharyngeal
apparatus in its final position;
g highly deformable pharynx
during its resynthesis, cuticular e f
valves (arrow) of the posterior
pharynx. asterisk salivary
glands; black arrow buccal ring;
ph pharynx. DIC (a–d, f, g);
PhC (e)

tardigradum and P. richtersi, the phases of the synthesis of
the cuticular structures of the buccal–pharyngeal apparatus
are similar between the two species and in agreement with
those schematized by Marcus (1936) for another eutardi-
grade, Macrobiotus hufelandi C.A.S. Schultze, 1834 and
briefly reported by Kristensen (1976) for the heterotardi-
grade Batillipes noerrevangi Kristensen, 1976. Also the
time needed for the complete resynthesis of the buccal–
pharyngeal apparatus is in agreement with the 24–48 h
reported by Crowe et al. (1971) for Paramacrobiotus
areolatus (Murray 1907) kept at 23°C. A step not yet well
understood is related to the repositioning of the complete
buccal–pharyngeal apparatus to its original position. While
the cuticular lining of the foregut connects and coalesces
with the body cuticle only at the end of the molting pro-
cess, the cuticular lining of the hindgut (proctodeum) is
always in continuity with the body cuticle and it is
discharged only with ecdysis. During the buccal–pharyn-
geal apparatus synthesis, the piercing stylets (that are
formed before the stylet coat) are the first structures that
can be morphologically discernible together with the buc-
cal tube. This is consistent with the fact that also during
embryonic development, the piercing stylets are the first
structures of the buccal–pharyngeal apparatus that can be
distinct within the embryo (Gabriel et al. 2007). This leads
us to suppose that both in the embryo and in the molting
adult the same pattern of development of the sclerified
structures of the buccal–pharyngeal apparatus occurs.
During their synthesis, the piercing stylets are formed
progressively backward from their anterior tips, both in
eutardigrades and in heterotardigrades (this study; Pollock
1970; Kristensen 1976), while the dissolution of the
piercing stylets during anoxic conditions (Bird and McC-
lure 1997) proceeds in the opposite direction.

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Zoomorphology (2012) 131:127–148
a e
b f
c g
d h
Fig. 17 EDX mapping of calcium and carbon in P. richtersi (a–d)
and M. cf. tardigradum (e–h) buccal–pharyngeal apparatuses.
a, e buccal–pharyngeal apparatus in toto (P. richtersi in ventral
view; M. cf. tardigradum in dorsal view, ESEM); b, f area in which
calcium (green) was detected; c overlapping of figures a and b (Ca in
red); g overlapping of figures e and f (Ca in red); d, h area in which
carbon (blue) was detected
Calcium carbonate encrustations
in the buccal–pharyngeal apparatus
Energy-dispersive X-ray analysis showed the presence of
calcium (Bird and McClure 1997; present study) in the
form of calcium carbonate (Bird and McClure 1997). The
presence of a stylet system with CaCO3 piercing stylets in
the feeding apparatus is an autoapomorphy of the tardi-
grades. Our finding of calcium, even though at relatively
low concentration, additionally in other structures of the
147
buccal–pharyngeal apparatus of P. richtersi, shows that
calcium carbonate incrustations are not an exclusive fea-
ture of heterotardigrades. Due to the dissolving of most of
the buccal tube, stylet and stylet support by weak acid,
Kristensen (1976) hypothesized that in the heterotardigrade
B. noerrevangi, these structures consist of CaCO3. In het-
erotardigrades, CaCO3 incrustations were reported, without
clear experimental evidence, at the level of the buccal tube
and the pharyngeal bars, as well as in the piercing stylets
(Marcus 1929; Kristensen 1987). Calcium carbonate has
the effect of stiffening and strengthening biological tissues;
therefore, the buccal–pharyngeal structures with CaCO3
are more rigid and robust for a more effective feeding
function. CaCO3 incrustations have been also reported for
bdelloid rotifer trophi (Melone et al. 1998) and buccal
structures of crustaceans (Hild et al. 2008). According to
Kristensen (1987), CaCO3 incrustations in the buccal–
pharyngeal apparatuses are plesiomorphic within hetero-
tardigrades considered having the highest number of
plesiomorphic characters comparing to eutardigrades.
Therefore, the presence of CaCO3 in the stylets, buccal
tube, and placoids of an eutardigrade species leads us to
suppose that these features were present in the ancestors of
both classes. The absence of calcium incrustation in the
buccal–pharyngeal apparatus of M. cf. tardigradum, and
presumably of all Apochela, is a derived condition.
The presence of calcium in the structures of the feeding
apparatus, the calcium carbonate nature of the piercing
stylet, the anatomical relationships of this structure with
the cuticular structures of the stylet system, and the
developmental sequence of the buccal–pharyngeal appa-
ratus resynthesis during the molting process obtained in
this study will help to elucidate the origin of the stylet
system characteristic of the phylum. Our findings further
illustrate that morphological fine-scale analysis accompa-
nied by comparative investigations give new understand-
ings of the structure and function of tardigrade anatomy
and adaptations, providing new characters for taxonomy
and for evolutionary study.
Acknowledgments The authors wish to thank Paul Bartels (Warren
Wilson College, Asheville, North Carolina, U.S.A.) for the great
courtesy of revising the English. The research is part of the project
MoDNA granted by Fondazione Cassa di Risparmio di Modena
(Italy) and the University of Modena and Reggio Emilia (Italy).
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