Hydrobiologia 432: 57–64, 2000.© 2000Kluwer Academic Publishers. Printed in the Netherlands.

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Functional morphology of the muscles inPhilodina sp.(Rotifera: Bdelloidea)

Rick Hochberg∗ & Marianne Klauser LitvaitisDepartment of Zoology and Center for Marine Biology, Rudman Hall, University of New Hampshire,Durham, NH 03824, U.S.A.Fax: +1-603-862-3784. E-mail: rickh@cisunix.unh.edu(∗author for correspondence)

Received 14 March 2000; accepted 21 April 2000

Key words:F-actin. phalloidin, fluorescent microscopy

Abstract

Whole-mounts ofPhilodinasp., a bdelloid rotifer, were stained with fluorescent-labeled phalloidin to visualize themusculature. Several different muscle types were identified including incomplete circular bands, coronal retractorsand foot retractors. Based on the position of the larger muscle bands in the body wall, their function during creepinglocomotion and tun formation was inferred. Bdelloid creeping begins with the contraction of incomplete circularmuscle bands against the hydrostatic pseudocoel, resulting in an anterior elongation of the body. One or moresets of ventral longitudinal muscles then contract bringing the rostrum into contact with the substrate, where itpresumably 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 These same longitudinal muscle sets are also used in ‘tun’ formation, in whichthe head and foot are withdrawn into the body. Three sets of longitudinal muscles supply the head region (anteriorhead segments) and function in withdrawal of the corona and rostrum. Two additional pairs of longitudinal musclesfunction to retract the anterior trunk segments immediately behind the head, and approximately five sets of longit-udinal retractors are involved in the withdrawal of the foot and posterior toes. To achieve a greater understandingof rotifer behavior, it is important to elucidate the structural complexity of body wall muscles in rotifers. The utilityof fluorescently-labeled phalloidin for the visualization of these muscles is discussed and placed in the context ofrotifer functional morphology.

Introduction

Most rotifers, whether parasitic, free-living, solitary,colonial, benthic, or planktonic, swim during somestage of their life cycle (Nogrady et al., 1993). Rotiferswimming is powered by the corona, a crown of ciliaat the anterior end that also functions in the productionof feeding currents. Metachronal waves pass along thecilia, setting up feeding currents in all rotifers and dir-ectional movements during planktonic locomotion infree-living species (Clément & Wurdak, 1991). Themorphology of the corona determines the type and ex-tent of locomotion in planktonic forms, and is also adiagnostic character in rotifer classification (Nogrady

et al., 1993). In some cases, rotifers may possess avery distinctive corona but rely on other means forlocomotion.

Rotifers of the order Bdelloidea (Class Digononta)are extremely abundant in lichens and mosses wherewater is generally in low abundance. Aqueous filmsaround these small plants presumably provide a suit-able medium for feeding and respiration; but rarely isthe water abundant enough to allow for long periodsof ciliary swimming. Instead, most micrometazoa usea form of crawling or creeping as the main form oflocomotion. Creeping movements in rotifers involvesa combination of adhesive glands and muscles to inch-worm across the substrate. The energy expenditure

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Figure 1. Fluorescence micrograph ofPhilodina sp. in a contracted, tun-like shape. A. Dorsal aspect ofPhilodina sp. B. Ventral aspect ofPhilodinasp. cm – circular muscle band, cr – coronal retractor, fr – foot retractor, hm – helical muscle, hr – head retractor, fr – foot retractors.× 630.

for this type of movement is unknown unlike that forswimming rotifers (Epp & Lewis, 1984).

Body movements in rotifers, produced by contrac-tion of the skeletal muscles, are generally not wellunderstood. Light microscopy has been useful in docu-menting rotifer muscle patterns because the body wallis highly transparent, but investigations concerning thefunction of specific muscle sets in a behavioral contextare limited (Amsellem & Clément, 1977, 1988; Clé-ment, 1987; Clément & Ansellem, 1989). Still, severalaspects of rotifer skeletal muscles have been describedin terms of striation patterns, sites of innervation andthe arrangement of motor units, and there is a generalunderstanding of how various muscles affect retrac-tion of the corona (Amsellem & Clément, 1977, 1988;Clément & Ansellem, 1989).

The purpose of this investigation was to describethe patterns of skeletal musculature in a bdelloid ro-tifer using a new fluorescent technique. Phallotoxins

linked to fluorescent dyes bind to the F-actin of musclecells allowing for the localization of large and smallmuscle fibers (Rieger et al., 1991). Similar stains havebeen employed to study the development of the cyto-skeleton inCaenorhabditis elegans(Priess & Hirsh,1986) and the muscle patterns of various turbellari-ans (Rieger et al., 1991, 1994; Tyler & Hyra, 1998;Hooge & Tyler, 1999a,b, 2000). Additionally, know-ledge of rotifer muscle patterns may provide clues toevolutionary relationships and phylogenetic trends inthe phylum as demonstrated for other animals (Hooge& Tyler, 1999b).

Materials and methods

Specimens ofPhilodinasp. Ehrenberg, 1830 were col-lected from a variety of moss and fructicose lichenin Durham, New Hampshire. Plants were placed in

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Figure 2. Dorsal view of slightly extendedPhilodina sp. fr – footretractor, ft – region of withdrawn foot and toes, hr – head retractor,mx – mastax, nr – neck retractor, td – trochal disc.× 630.

small dishes of spring water and allowed to stand forseveral days. The plants were then removed from thedishes and individual rotifers were removed from thebowls with a micropipette. Rotifers were relaxed in1% MgCl2 for 1 h before processing. In most cases,rotifers contracted upon introduction into MgCl2 mak-

Figure 3. Ventrolateral view ofPhilodina sp. revealing incompletecircular muscle bands. cm – circular muscles, cmg – ventral gapbetween insertion sites of a circular muscle, m – mouth.× 630.

ing species identification impossible. Animals werefixed in small finger-bowls in 4% formaldehyde in0.01 M PBS for 1 h followed by a 5 min buffer rinse.Permeabilization in 0.1% Triton-X in 0.01M PBS for1 h was followed by staining with Alexa 488 phal-loidin (Molecular Probes, Eugene OR) for 40 min.Stained specimens were rinsed briefly with 0.01 M

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PBS, transferred to a microscope slide, and mountedwith Gel/Mount. Control specimens were fixed andmounted without stain. In several instances, contrac-ted rotifers failed to stain, so permeabilization timewas increased to 2 h and staining to 1.5 h. All spe-cimens were kept at 4◦C for several days beforeviewing with a Zeiss Axiophot epifluorescence mi-croscope equipped with a SPOT Cooled Color digitalcamera (Diagnostic Instruments, Inc.).

Results

Phalloidin staining of F-actin inPhilodinasp. revealeda complex array of individual muscle bands (Figures1–5), allowing for mapping of their orientation andsubsequent functional interpretation (Figure 7). Thebody wall musculature consists of two major groups:thick, outer circular bands and thin, inner longitudinalfibers. Circular muscles were present from the headregion around the corona to the foot. Circular bandsin the head and foot region were thinner in diameterthan those of the trunk. Trunk circular muscles rangedbetween 8 and 10µm in diameter; head and footmuscles were 4–6µm in diameter. There were approx-imately 14–16 circular muscle bands present in a 1000µm long specimen. Unfortunately, most specimenswere contracted to some degree making it difficult toquantify the exact number of circular bands. All cir-cular muscles appeared incomplete, only forming arcsinstead of complete bands (Figure 3). The insertionpoints of these arcs were separated by 10–20µm, andappeared incomplete on the ventral aspect.

A large network of longitudinal muscles waspresent beneath the circular muscle bands. Musclesappeared to be oriented in two functional series: an-terior retractors and posterior retractors. Most lon-gitudinal muscle bands were approximately 1–3µmin diameter. Several longitudinal retractors appearedto span the length of an animal (Figures 1–3). Mostoriginated just below the rostrum and inserted pos-teriorly at the base of the trunk or in the foot. Nomuscle fibers were observed in the spurs. The head(=coronal/rostral) region was supplied with three setsof longitudinal muscles. The most medial pair insertedventrally at the top of the head, and posteriorly, bi-furcated at approximately 40% body length, insertingclose to the ventral midline (Figure 2). The middle pairbifurcated at the anterior end and inserted posteriorlyon the lateral body wall at approximately 40% body

length. The most lateral pair inserted posteriorly in thefoot.

Dorsally, a single pair of longitudinal bands sup-plied the upper neck segments (just below the head).The pair spanned the length of an animal, insertinganteriorly in the neck region and posteriorly at thebase of the trunk. Immediately ventral to this pairof muscles, was a longitudinal band with a three-branched head inserting close to the dorsal muscle,anteriorly (Figure 1) and posteriorly (Figure 4). Asecond pair of ventral neck muscles also supplied thelateral neck region, originating close to the ventralmidline at approximately 50% body length.

Several longitudinal muscle bands supplied thefoot and were implicated in the telescopic retraction ofthe foot into the body proper. A single pair of dorsalbands inserted inside the foot close to the base. Vent-rally, at least four pairs of muscles also supplied thefoot, only a single pair of which spanned the lengthof an animal to insert in the head region. The anteriorportion of the other three muscle pairs inserted closeto mid-body length.

Several actin-containing fibers with a distinctlydifferent morphology and/or orientation from thetypical circular and longitudinal muscles were alsopresent. One pair of fibers formed a helix close tothe mastax (Figures 1 and 5). These fibers appearedto insert on large longitudinal bands, but this couldnot be confirmed. Several other actin-containing bandscoursed through the head and foot regions of numer-ous specimens. (Figure 6).

Discussion

Philodina sp. is a well-known bdelloid rotifer fromfreshwater and semi-terrestrial environments where itoften inhabits various mosses and lichens. Severalaspects ofPhilodinamorphology have been well doc-umented including the ultrastructure of the coronalcilia, cerebral eyes, skeletal lamina, pedal glands,skeletal muscles (literature in Clément & Wurdak,1991) and morphological changes during anydrobi-osis (Dickson & Mercer, 1967). Clément & Amsellem(1989) described the ultrastructure of skeletal musclesin rotifers and found a variety of striation patterns.Skeletal muscles are either cross or obliquely striated,or smooth in appearance. The muscles ofPhilodinaroseolaare either smooth or obliquely striated depend-ing on their orientation (Clément & Amsellem, 1989).The longitudinal retractor muscles are obliquely stri-

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Figure 4. Dorsal view of the posterior end of a contractedPhilodina sp. The foot is withdrawn anteriorly. vr – ventral retractor, dr – dorsalretractor, ft – foot.× 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(Clément & Amsellem, 1989) and are likely to be ad-aptations to predation, functioning predominantly inescape responses.

Locomotion inPhilodina sp. and most bdelloidsresembles leech-like or inch-worm type movements,and involves a series of body contractions and elonga-tions that utilize both circular and longitudinal mus-culature (Figure 8). Creeping begins with a generalelongation of the body created by contraction of thelarge circular muscle bands. The hydrostatic pres-sure of the pseudocoel probably serves as an ant-agonist during this contraction, resulting in generalbody extension. Next, the animal contracts its vent-ral longitudinal musculature to bring the rostrum incontact with the substrate. The precise nature of theadhesive attachment is unknown, but may involvesecretions of the retrocerebral apparatus, a muscle-wrapped exocrine gland present only in species thatexhibit creeping-type movement patterns (Clément &Wurdak, 1991). Following attachment of the anterior

end, the trunk and foot are pulled forward via contrac-tion of several sets of longitudinal retractor muscles,shortening the body to approximately 50% normalbody length. While a portion of the foot appears totelescope into the trunk (Figure 2), much of the foot

Figure 5. Helical fibers in the vicinity of the mastax (not seen). hf– helical fibers, cm – circular muscle.× 1000.

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Figure 6. Ventrolateral view of the anterior end ofPhilodina sp. revealing small diameter actin-containing fibers. arrows – actin fibers ofunknown function.× 630.

is swept underneath the dorsally arched trunk.Philod-ina sp. is highly flexible and the syncytial integumentdoes not appear to inhibit body compression. Next,the toes of the foot are attached to the substrate viasecretions of the pedal glands. Elongation of the bodythen resumes with contraction of the circular muscles.This movement often alternates with bouts of ciliarylocomotion via the corona.

Withdrawal behavior in a sessile bdelloid involvesretraction of the corona and general body contractioninto a tun-like shape. The same condition is seen inanhydrobiotic rotifers, wherein tun formation precedeshabitat desiccation, resulting in dry rotifers with lowmetabolic water and high tolerance to environmentalextremes (Nogrady et al., 1993). The formation of thiscontracted, protective state was performed by the samelongitudinal muscles used in forward creeping, onlythe contraction was more extensive.The musculatureof Philodina sp. corresponds closely to that knownfor other bdelloid rotifers likeRotaria sp. (Hyman,1951). Both genera contain well developed circular

and longitudinal muscle bands. Interestingly, the cir-cular bands in both genera are incomplete, and formdorsal arcs instead of complete circles. This appearsto be a common phenomenon among rotifers, foundalso in members of the order Ploimida (Hyman, 1951).However, inRotaria, the circular bands bifurcate be-fore terminating (Hyman, 1951) whereas inPhilodinasp. and the ploimids the terminal insertions do notappear to branch. The functional difference betweena branched and unbranched insertion is unknown, asis the difference between a complete and incompletemuscle band, but based on behavioral observations ofrotifer locomotion, the results appear to be similar.

In addition to the large skeletal muscles inPhilod-ina, several smaller F-actin containing fibers were alsorevealed. These fibers had a small diameter and weremost apparent in the head and foot region. Basedon their position, the head fibers may be implicatedin some aspect of coronal coordination or perhapsin supplying the mastax. The function of the footfibers is unknown. The precise nature of these small

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Figure 7. Composite drawing (dorsal and ventral) of the longit-udinal musculature of a contractedPhilodina sp. Muscles used inwithdrawal of the foot are colored.

diameter fibers remains undetermined, but based ontheir orientation and morphology, they may be neural.Fluorescently-labeled phalloidin has been shown to la-bel F-actin in vertebrate neurons (Barber et al., 1996),although not in invertebrates where the stain has beenapplied (Priess & Hirsh, 1986; Tyler & Hyra, 1998;Hooge & Tyler, 1999a,b, 2000). A complete ultra-structural analysis is necessary before confirming theidentity of these fibers.

Although fluorescent visualization of the rotifermuscular system provides greater advantages thanbrightfield microscopy, making the rotifer muscularsystem easier to identify relative to brightfield micro-scopy, several difficulties inherent in rotifer fixationremain. Most bdelloid rotifers contract into tuns uponintroduction to either anaesthetic or fixative (Wal-lace & Snell, 1991), resulting in partially shriveledspecimens and/or poor labeling of F-actin containing

Figure 8. Sequence of creeping movements ofPhilodina sp. andthe muscles involved. (A) Contraction of circular muscles causesforward elongation of the anterior end. (B) Contraction of the vent-ral longitundal muscles results in ventral flexion and contact of therostrum with the substrate. (C) The foot and toes are pulled towardthe trunk via contraction of foot retractors.

tissue. In partially contracted individuals, all muscleswere stained, but insertion points remained difficultto identify. In specimens that contracted into tuns,no staining was seen with standard permeabilizationand staining, necessitating an increase in time. It isunknown 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 musclesof the bdelloid rotiferPhilodina sp. were studiedusing fluorescently-labeled phalloidin. A variety ofmuscle bands were observed, including large diameter,incomplete circular bands and thinner longitudinalfibers. Based on the orientation of these muscles, theirroles in creeping locomotion and tun formation were

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discussed. The circular muscles function in body ex-tension, contracting against the hydrostatic pressure ofthe pseudocoel, and causing body elongation in an an-terior direction. The longitudinal muscles function asretractors, pulling the trunk and foot forward, resultingin a leech-like movement. Tun formation appears toinvolve only the longitudinal retractor muscles.

Philodinais but one genus of the order Bdelloideathat contains a large variety of creeping forms. In suchspecies, the muscles play a prominent role in benthiclocomotion, as opposed to semi-pelagic species thatlive in the water column and rely heavily on swimmingusing coronal cilia. It therefore seems likely that par-ticular muscles sets in bdelloids, particularly the footretractors, will be better developed than in planktonicspecies. The validity of this hypothesis is undeter-mined but should be easy to verify with the expandeduse of the presented phalloidin protocol. In addition,the use of this stain will allow biologists to gain furtherinsight into the evolution of different rotifer clades byunderstanding how muscle development may changewith changes in habitats (interstitial, epiphytic, plank-tonic, parasitic) and changes in morphology duringcyclomorphosis.

Acknowledgements

This study was funded by grants from the GraduateSchool, Department of Zoology, Center for MarineBiology and by the Hubbard Marine Research Initit-ation Program at the University of New Hampshire.

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