Functional morphology of the muscles in Philodina sp. (Rotifera

Hydrobiologia 432: 57-64,2000.
0 2000 Kluwer Academic Publishers. Printed in the Netherlands.
Functional morphology of the muscles in Philodina sp.
(Rotifera: Bdelloidea)
Rick Hochberg* & Marianne Klauser Litvaitis
Department of Zoology and Centerfor Marine Biology, Rudman Hall, University of New Hampshire,
Durham, NH 03824, U.S.A.
Fax: +1-603-862-3784.E-mail: [email protected]
("'authorfor correspondence)
Received 14 March 2000. accented 21 Aoril2000
Key words: F-actin. phalloidin, fluorescent microscopy
Abstract
Whole-mounts of Philodina sp., a bdelloid rotifer, were stained with fluorescent-labeledphalloidin to visualize the
musculature. Several different muscle types were identified including incomplete circular bands, coronal retractors
and foot retractors. Based on the position of the larger muscle bands in the body wall, their function during creeping
locomotion and tun formation was inferred. Bdelloid creeping begins with the contraction of incomplete circular
muscle bands against the hydrostatic pseudocoel, resulting in an anterior elongation of the body. One or more
sets of ventral longitudinal muscles then contract bringing the rostrum into contact with the substrate, where it
presumably attaches via adhesive glands. Different sets of ventral longitudinal muscles, foot and trunk retractors,
function to pull the body forward. These same longitudinal muscle sets are also used in 'tun' formation, in which
the head and foot are withdrawn into the body. Three sets of longitudinal muscles supply the head region (anterior
head segments) and function in withdrawal of the corona and rostrum. Two additional pairs of longitudinal muscles
function to retract the anterior trunk segments immediately behind the head, and approximately five sets of longitudinal retractors are involved in the withdrawal of the foot and posterior toes. To achieve a greater understanding
of rotifer behavior, it is important to elucidate the structural complexity of body wall muscles in rotifers. The utility
of fluorescently-labeledphalloidin for the visualization of these muscles is discussed and placed in the context of
rotifer functional morphology.
Introduction
Most rotifers, whether parasitic, free-living, solitary,
colonial, benthic, or planktonic, swim during some
stage of their life cycle (Nogrady et al., 1993). Rotifer
swimming is powered by the corona, a crown of cilia
at the anterior end that also functions in the production
of feeding currents. Metachronal waves pass along the
cilia, setting up feeding currents in all rotifers and directional movements during planktonic locomotion in
free-living species (Clement & Wurdak, 1991). The
morphology of the corona determines the type and extent of locomotion in planktonic forms, and is also a
diagnostic character in rotifer classification (Nogrady
et al., 1993). In some cases, rotifers may possess a
very distinctive corona but rely on other means for
locomotion.
Rotifers of the order Bdelloidea (Class Digononta)
are extremely abundant in lichens and mosses where
water is generally in low abundance. Aqueous films
around these small plants presumably provide a suitable medium for feeding and respiration; but rarely is
the water abundant enough to allow for long periods
of ciliary swimming. Instead, most micrometazoa use
a form of crawling or creeping as the main form of
locomotion. Creeping movements in rotifers involves
a combination of adhesive glands and muscles to inchworm across the substrate. The energy expenditure
Figure I . Fluorescs
. B. Ventral aspect of
ograph of Philodina sp. in a contracted, tun-like shape. A. Dorsal aspect of Phi
Philodina sp. cm -circular muscle band, cr - coronal retractor, fr - foot retractor, hm - helical muscle, hr - head retractor, fr - foot retractors.
x 630.
for this type of movement is unknown unlike that for
swimming rotifers (Epp & Lewis, 1984).
Body movements in rotifers, produced by contraction of the skeletal muscles, are generally not well
understood. Light microscopy has been useful in documenting rotifer muscle patterns because the body wall
is highly transparent, but investigationsconcerning the
function of specific muscle sets in a behavioral context
are limited (Amsellem & Clement, 1977, 1988; Clement, 1987; Clement & Ansellem, 1989). Still, several
aspects of rotifer skeletal muscles have been described
in terms of striation patterns, sites of innervation and
the arrangement of motor units, and there is a general
understanding of how various muscles affect retraction of the corona (Amsellem & Clement, 1977,1988;
Clement & Ansellem, 1989).
The purpose of this investigation was to describe
the patterns of skeletal musculature in a bdelloid rotifer using a new fluorescent technique. Phallotoxins
linked to fluorescent dyes bind to the F-actin of muscle
cells allowing for the localization of large and small
muscle fibers (Rieger et al., 1991). Similar stains have
been employed to study the development of the cytoskeleton in Caenorhabditis elegans (Priess & Hirsh,
1986) and the muscle patterns of various turbellarians (Rieger et al., 1991, 1994; Tyler & Hyra, 1998;
Hooge & Tyler, 1999a,b, 2000). Additionally, knowledge of rotifer muscle patterns may provide clues to
evolutionary relationships and phylogenetic trends in
the phylum as demonstrated for other animals (Hooge
& Tyler, 1999b).
Materials and methods
Specimens of Philodina sp. Ehrenberg, 1830 were collected from a variety of moss and fructicose lichen
in Durham, New Hampshire. Plants were placed in
Figure 3. Ventrolateral view of I'l~ilotliiui sp. revealing incon~plete
circular muscle bands. cm - circular muscles, cmg ventral gap
between insertion si~esof ;I circular muscle. I N - mouth, x 630.
-
Figure 2. Dorsal view of slightly extended Philodina sp. fr - foot
retractor, ft - region of withdrawn foot and toes, hr - head retractor,
mx - mastax, nr - neck retractor, td - trochal disc. x 630.
small dishes of spring water and allowed to stand for
several days. The plants were then removed from the
dishes and individual rotifers were removed from the
bowls with a micropipette. Rotifers were relaxed in
1% MgCl2 for 1 h before processing. In most cases,
rotifers contracted upon introduction into MgCl2 mak-
ing species identification impossible. Animals were
fixed in small finger-bowls in 4% formaldehyde in
0.01 M PBS for 1 h followed by a 5 min buffer rinse.
Permeabilization in 0.1% Triton-X in 0.01M PBS for
1 h was followed by staining with Alexa 488 phalloidin (Molecular Probes, Eugene OR) for 40 min.
Stained specimens were rinsed briefly with 0.01 M
PBS, transferred to a microscope slide, and mounted
with Gel/Mount. Control specimens were fixed and
mounted without stain. In several instances, contracted rotifers failed to stain, so permeabilization time
was increased to 2 h and staining to 1.5 h. All specimens were kept at 4 OC for several days before
viewing with a Zeiss Axiophot epifluorescence microscope equipped with a SPOT Cooled Color digital
camera (Diagnostic Instruments, Inc.).
Results
Phalloidin staining of F-actin in Philodina sp. revealed
a complex array of individual muscle bands (Figures
1-5), allowing for mapping of their orientation and
subsequent functional interpretation (Figure 7). The
body wall musculature consists of two major groups:
thick, outer circular bands and thin, inner longitudinal
fibers. Circular muscles were present from the head
region around the corona to the foot. Circular bands
in the head and foot region were thinner in diameter
than those of the trunk. Trunk circular muscles ranged
between 8 and 10 p m in diameter; head and foot
muscles were 4-6 p m in diameter. There were approximately 14-16 circular muscle bands present in a 1000
p m long specimen. Unfortunately, most specimens
were contracted to some degree making it difficult to
quantify the exact number of circular bands. All circular muscles appeared incomplete, only forming arcs
instead of complete bands (Figure 3). The insertion
points of these arcs were separated by 10-20 pm, and
appeared incomplete on the ventral aspect.
A large network of longitudinal muscles was
present beneath the circular muscle bands. Muscles
appeared to be oriented in two functional series: anterior retractors and posterior retractors. Most longitudinal muscle bands were approximately 1-3 p m
in diameter. Several longitudinal retractors appeared
to span the length of an animal (Figures 1-3). Most
originated just below the rostrum and inserted posteriorly at the base of the trunk or in the foot. No
muscle fibers were observed in the spurs. The head
(=coronal/rostral) region was supplied with three sets
of longitudinal muscles. The most medial pair inserted
ventrally at the top of the head, and posteriorly, bifurcated at approximately 40% body length, inserting
close to the ventral midline (Figure 2). The middle pair
bifurcated at the anterior end and inserted posteriorly
on the lateral body wall at approximately 40% body
length. The most lateral pair inserted posteriorly in the
foot.
Dorsally, a single pair of longitudinal bands supplied the upper neck segments (just below the head).
The pair spanned the length of an animal, inserting
anteriorly in the neck region and posteriorly at the
base of the trunk. Immediately ventral to this pair
of muscles, was a longitudinal band with a threebranched head inserting close to the dorsal muscle,
anteriorly (Figure 1) and posteriorly (Figure 4). A
second pair of ventral neck muscles also supplied the
lateral neck region, originating close to the ventral
midline at approximately 50% body length.
Several longitudinal muscle bands supplied the
foot and were implicated in the telescopic retraction of
the foot into the body proper. A single pair of dorsal
bands inserted inside the foot close to the base. Ventrally, at least four pairs of muscles also supplied the
foot, only a single pair of which spanned the length
of an animal to insert in the head region. The anterior
portion of the other three muscle pairs inserted close
to mid-body length.
Several actin-containing fibers with a distinctly
different morphology and/or orientation from the
typical circular and longitudinal muscles were also
present. One pair of fibers formed a helix close to
the mastax (Figures 1 and 5). These fibers appeared
to insert on large longitudinal bands, but this could
not be confirmed. Several other actin-containing bands
coursed through the head and foot regions of numerous specimens. (Figure 6).
Discussion
Philodina sp. is a well-known bdelloid rotifer from
freshwater and semi-terrestrial environments where it
often inhabits various mosses and lichens. Several
aspects of Philodina morphology have been well documented including the ultrastructure of the coronal
cilia, cerebral eyes, skeletal lamina, pedal glands,
skeletal muscles (literature in Clement & Wurdak,
1991) and morphological changes during anydrobiosis (Dickson & Mercer, 1967). Clement & Amsellem
(1989) described the ultrastructure of skeletal muscles
in rotifers and found a variety of striation patterns.
Skeletal muscles are either cross or obliquely striated,
or smooth in appearance. The muscles of Philodina
roseola are either smooth or obliquely striated depending on their orientation (Clement & Amsellem, 1989).
The longitudinal retractor muscles are obliquely stri-
Figure 4. Dorsal view of the posterior end of a contracted Philodina sp. The foot is withdrawn anteriorly. w - ventral retractor, dr - dorsal
retractor, ft - foot x 1000.
ated and function in the withdrawal of the corona,
contraction of the body and general body flexion.
These muscles have the characteristics of fast muscle
(Clement & Amsellem, 1989) and are likely to be adaptations to predation, functioning predominantly in
escape responses.
Locomotion in Philodina sp. and most bdelloids
resembles leech-like or inch-worm type movements,
and involves a series of body contractions and elongations that utilize both circular and longitudinal musculature (Figure 8). Creeping begins with a general
elongation of the body created by contraction of the
large circular muscle bands. The hydrostatic pressure of the pseudocoel probably serves as an antagonist during this contraction, resulting in general
body extension. Next. the animal contracts its ventral longitudinal musculature to bring the rostrum in
contact with the substrate. The precise nature of the
adhesive attachment is unknown, but may involve
secretions of the retrocerebral apparatus, a musclewrapped exocrine gland present only in species that
exhibit creeping-type movement patterns (Clement &
Wurdak, 1991). Following attachment of the anterior
end, the trunk and foot are pulled forward via contraction of several sets of longitudinal retractor muscles,
shortening the body to approximately 50% normal
body length. While a portion of the foot appears to
telescope into the trunk (Figure 2), much of the foot
Figure 5. Helical fibers in the vicinity of the mastax (not seen). hf
fibers, cm -circular muscle. x 1000.
- helical
Figure 6. Ventrolateral view of the anterior end of Philodina sp. revealing small diameter actin-containing fibers. arrows - actin fibers of
unknown function. x 630.
is swept underneath the dorsally arched trunk. Philodina sp. is highly flexible and the syncytial integument
does not appear to inhibit body compression. Next,
the toes of the foot are attached to the substrate via
secretions of the pedal glands. Elongation of the body
then resumes with contraction of the circular muscles.
This movement often alternates with bouts of ciliary
locomotion via the corona.
Withdrawal behavior in a sessile bdelloid involves
retraction of the corona and general body contraction
into a tun-like shape. The same condition is seen in
anhydrobioticrotifers, wherein tun formation precedes
habitat desiccation, resulting in dry rotifers with low
metabolic water and high tolerance to environmental
extremes (Nogrady et al., 1993). The formation of this
contracted, protective state was performed by the same
longitudinal muscles used in forward creeping, only
the contraction was more extensive.The musculature
of Philodina sp. corresponds closely to that known
for other bdelloid rotifers like Rotaria sp. (Hyman,
1951). Both genera contain well developed circular
and longitudinal muscle bands. Interestingly, the circular bands in both genera are incomplete, and form
dorsal arcs instead of complete circles. This appears
to be a common phenomenon among rotifers, found
also in members of the order Ploimida (Hyman, 1951).
However, in Rotaria, the circular bands bifurcate before terminating (Hyman, 1951) whereas in Philodina
sp. and the ploimids the terminal insertions do not
appear to branch. The functional difference between
a branched and unbranched insertion is unknown, as
is the difference between a complete and incomplete
muscle band, but based on behavioral observations of
rotifer locomotion, the results appear to be similar.
In addition to the large skeletal muscles in Philodina, several smaller F-actin containing fibers were also
revealed. These fibers had a small diameter and were
most apparent in the head and foot region. Based
on their position, the head fibers may be implicated
in some aspect of coronal coordination or perhaps
in supplying the mastax. The function of the foot
fibers is unknown. The precise nature of these small
B
c
Figure 7. Composite drawing (dorsal and ventral) of the longitudinal musculature of a contracted Philodina sp. Muscles used in
withdrawal of the foot are colored.
diameter fibers remains undetermined, but based on
their orientation and morphology, they may be neural.
Fluorescently-labeledphalloidin has been shown to label F-actin in vertebrate neurons (Barber et al., 1996),
although not in invertebrates where the stain has been
applied (Priess & Hirsh, 1986; Tyler & Hyra, 1998;
Hooge & Tyler, 1999a,b, 2000). A complete ultrastructural analysis is necessary before confirming the
identity of these fibers.
Although fluorescent visualization of the rotifer
muscular system provides greater advantages than
brightfield microscopy, making the rotifer muscular
system easier to identify relative to brightfield microscopy, several difficulties inherent in rotifer fixation
remain. Most bdelloid rotifers contract into tuns upon
introduction to either anaesthetic or fixative (Wallace & Snell, 1991), resulting in partially shriveled
specimens and/or poor labeling of F-actin containing
Figure 8. Sequence of creeping movements of Philodina sp. and
the muscles involved. (A) Contraction of circular muscles causes
forward elongation of the anterior end. (B) Contraction of the ventral longitundal muscles results in ventral flexion and contact of the
rostrum with the substrate. (c) ~h~ foot and toes are
the trunk via contraction of foot retractors.
tissue. In partially contracted individuals, all muscles
were stained, but insertion points remained difficult
to identify. In specimens that contracted into tuns,
no staining was seen with standard permeabilization
and staining, necessitating an increase in time. It is
unknown whether rotifers incapable of forming tuns
(Monogononta, Seissonoidea; Wallace & Snell, 1991)
can also prevent staining when contracted.
Conclusions
The distribution of F-actin filaments in the muscles
of the bdelloid rotifer Philodina sp. were studied
using fluorescently-labeled phalloidin. A variety of
muscle bands were observed, including large diameter,
incomplete circular bands and thinner longitudinal
fibers. Based on the orientation of these muscles, their
roles in creeping locomotion and tun formation were
discussed. The circular muscles function in body extension, contracting against the hydrostatic pressure of
the pseudocoel, and causing body elongation in an anterior direction. The longitudinal muscles function as
retractors, pulling the trunk and foot forward, resulting
in a leech-like movement. l k n formation appears to
involve only the longitudinal retractor muscles.
Philodina is but one genus of the order Bdelloidea
that contains a large variety of creeping forms. In such
species, the muscles play a prominent role in benthic
locomotion, as opposed to semi-pelagic species that
live in the water column and rely heavily on swimming
using coronal cilia. It therefore seems likely that particular muscles sets in bdelloids, particularly the foot
retractors, will be better developed than in planktonic
species. The validity of this hypothesis is undetermined but should be easy to verify with the expanded
use of the presented phalloidin protocol. In addition,
the use of this stain will allow biologists to gain further
insight into the evolution of different rotifer clades by
understanding how muscle development may change
with changes in habitats (interstitial, epiphytic, planktonic, parasitic) and changes in morphology during
cyclomorphosis.
Acknowledgements
This study was funded by grants from the Graduate
School, Department of Zoology, Center for Marine
Biology and by the Hubbard Marine Research Inititation Program at the University of New Hampshire.
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