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Muscle organization of the cubozoan jellyfish Tripedalia cystophora Conant 1897.

Introduction

Cubomedusae are remarkable among cnidarians for their complex, lensed eyes and the rapidity and accuracy of their swimming adjustments in response to visual stimuli. A great deal of attention has been paid to cubomedusan visual abilities, most centering on behavioral reactions to optical stimuli or the physical properties of the lens and retina (Martin, 2002; Coates, 2003; Nilsson et al., 2005). Each of their four rhopalia have two lensed, camera-type eyes that focus images in the vicinity of a photoreceptor layer (Nilsson, 1990; Nilsson et al., 2005). The optical properties of the lens make it likely that images are focused without significant spherical aberration (Land, 1990). Behaviorally, dark-adapted cubomedusae swim toward a dim light and can distinguish between light and dark objects (swim toward the former and away from the latter) when the objects are as small as 1-cm wide (Matsumoto, 1995). Chironex fleckeri was shown to respond behaviorally to 1 cm objects from a distance of 50 cm (Hamner et al., 1995). Convincing evidence suggests that cubomedusan eyes are capable of color and spatial discrimination (Theobald and Coates, 2004).

Strong, efficient swimmers (Stewart, 1996), cubomedusae can change swim direction in one or two individual bell contractions. Turns are produced through asymmetrical contractions of the velarium, a circular muscle ring that projects at right angles from the walls of the bell margin (Gladfelter, 1973). The velarium narrows the bell opening in a nozzle-like fashion during the swim contractions.

The velarium is analogous, but not homologous, to the velum of hydrozoan medusae. Four morphological features set the cubomedusan velarium apart from the hydrozoan velum and suggest that the velarium is derived from scyphozoan-like marginal lobes (Conant, 1898; Gladfelter, 1973): (1) the velarium is penetrated by gastric canals (never in the velum); (2) swim musculature of the velarium is continuous with that of the subumbrella; (3) the velarium, unlike the velum, does not possess exumbrellar radial muscle; (4) four bracket-like frenula support the velarium in the perradii.

Some hydromedusae are as maneuverable as cubomedusae. The hydromedusan velum includes both striated, circular muscle (subumbrellar) and radial, smooth muscle (exumbrellar), so any deformation of the velar ring to form a nozzle can be accomplished with regional (asymmetric) contraction of the radial musculature. In cubomedusae, velarial muscle fibers are all circular and striated, yet the velarium clearly forms a directional nozzle during turning (Gladfelter, 1973). This suggests that asymmetric contractions of the subumbrella and velarium (including frenula) must involve regionally enhanced contractions of the swim musculature. Frequency-dependent facilitation has been described in the swim muscle of Carybdea, as well as apparently asymmetric enhanced contractions in response to quick "double pulses" in the motor nerve net (Satterlie, 1979, 2002).

In our investigation of the neural and muscular organization of the swim system of the cubomedusa Tripedalia cystophora Conant, 1897, our attention was drawn to the four velarial frenula--buttress-like muscular brackets that brace the right-angle connection between the velarium and subumbrella in the perradii. Initially, the frenula were thought to be passive structural reinforcements, but upon closer examination, the circular musculature of the velarium appeared to turn 90 degrees, in a radial direction, on each face of the frenula. This, together with immunohistochemical data, suggests that the musculature of the frenula receives the highest density innervation of the motor nerve net (Coates and Satterlie, in prep.), prompting a reexamination of the muscular organization of Tripedalia.

Materials and Methods

Specimens of Tripedalia cystophora were obtained from a culture at Hopkins Marine Station, Pacific Grove, California, courtesy of Melissa M. Coates. Prior to use, animals were held in artificial seawater at room temperature (20-22[degrees]C).

Before fixation with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4), the animals were relaxed in a 1:2 mixture of isotonic Mg[Cl.sub.2] (0.33 M) and seawater. To minimize precipitate formation, the preparations were rinsed with seawater once quickly prior to immersion in fixative. Tissue pieces were fixed for 4-12 h (time was determined mostly by convenience and no differences were noted).

Fixed material was washed at least four times in 0.1 M phosphate buffer (pH 7.4) containing 0.01% Triton X-100 or 0.05% Tween 20. The tissue was dehydrated and then rehydrated through a graded ethanol series to further permeabilize the samples. After blocking in 5% goat serum in phosphate buffer (2-4 h), the tissue was incubated for 24-48 h in a 1:200 dilution of anti-actin antibody (Sigma #A5060), washed in phosphate buffer (4-8 changes), and incubated for 12-24 h in fluorescent-tagged secondary antibody (Molecular Probes, goat anti-rabbit Alexa Fluor 488 or Alexa Fluor 568). After another wash series in phosphate buffer, tissue pieces were cleared and then mounted in a 9:1 mixture of glycerol and 50 mM Tris buffer (pH 9).

Alternately, tissue pieces were fixed, washed, and permeabilized as above, and then incubated in a 1:200 dilution of Alexa Fluor 568 phalloidin (Molecular Probes product A-12380) overnight; followed by washing, clearing, and mounting.

Results from the anti-actin and phalloidin preparations were identical in terms of details of muscle organization. However, the anti-actin technique gave a better signal-to-noise ratio and produced better photographs. Therefore, all of the figures are from anti-actin immunohistochemical preparations.

Preparations were viewed in a Nikon epifluorescence microscope with FITC or TRITC filter cubes, and photographed with a SPOT digital camera.

Results

Subumbrellar organization

The subumbrella of Tripedalia cystophora is divided into quadrants bounded by the interradii, each marked by the emergence of a common tentacle pedalium at the bell margin, giving the medusa its cube-like shape (Fig. 1). Each pedalium divides into three blades, and each blade gives rise to a single, unbranching tentacle.

Each quadrant is radially subdivided at the perradius, midway between the interradii. At each of the four perradii, a rhopalium hangs in an exumbrellar concavity (niche), a short distance up from the margin. The rhopalium is connected to the subumbrellar tissue by a rhopalial stalk that runs through to the subumbrella. Neuronal networks run through the rhopalial stalk and connect with the subumbrellar nerve ring. Each rhopalium has two complex, lensed eyes, four simple ocelli, and a statolith.

A nerve ring runs, at an oblique angle, from the common base of the three pedalia (at the interradius) to the origin of the rhopalial stalk, then back down to the next pedalial base, and so on around the subumbrella (Fig. 1). The nerve ring is nearest, but not at, the bell margin at the pedalial base and loops upward at the rhopalial stalk, well above the margin.

At the edge of the bell, a shelf of muscular tissue bends inward, at a near-right angle in relaxed animals (Fig. 1). This velarium narrows the bell opening during a swim contraction and thus serves the same function as the velum of hydromedusae (Gladfelter, 1973). The structure of the velarium is not remarkable at the interradii, but it is supported by a buttress-like, subumbrellar frenulum at each perradius (Fig. 1). The frenula are triangular muscular brackets that run in a radial direction from the velarium, tapering near the level of the center of the rhopalial niche. In a radial plane, the frenula are narrow and flat. Viewed from the side, they are as wide as the velarium at the site of velarial attachment, and they narrow nearly to a point at the site of subumbrellar insertion.

At the subumbrellar apex, a four-lobed manubrium hangs within the bell (not shown in Fig. 1). A narrow manubrial stalk flares into a bulbous body that continues as four slightly elongated lips, forming the mouth opening. The lips are longer in the perradial plane, giving the mouth a scalloped appearance.

Swim musculature

The bulk of the subumbrellar musculature is circular in orientation and striated (Fig. 2), lining nearly the entire subumbrellar cavity and the subumbrellar side of the velarium. This musculature provides the main motive force for ejecting water from the subumbrellar cavity during swimming and for narrowing the velarium to form a nozzle with a restricted diameter.

In the subumbrella proper (excluding the velarium), the striated muscle sheet is interrupted by two types of structures. Muscle fibers terminate on both sides of the nerve ring and do not cross over or under it (Fig. 3).

At the perradii, raised bands of smooth muscle run radially, from just below the nerve ring, upward into the manubrial stalk (described below). The adjacent sheets of striated swim muscle terminate at the edges of the perradial muscle band, crossing neither over nor under it.

At the interradii, the tissue sheets are connected to the bell mesoglea, but the circular, striated muscle sheets are continuous, showing no interruptions (Fig. 3). The striated nature of the swim musculature was verified at high magnification of the anti-actin immunohistochemical (actin-IH) preparations (Fig. 2).

The circular musculature of the velarium is continuous with that of the subumbrella below the level of the nerve ring, and is indistinguishable from it. The muscle fibers show striations in actin-IH preparations. The velarial musculature forms a ring that is interrupted at the perradii, but not in the same fashion as in the subumbrella.

A frenulum interrupts the ring of tissue of the velarium, but this interruption does not extend through to the exumbrella. The frenulum represents an inward and upward fold of subumbrellar tissue that forms a double muscle layer separated by a narrow wedge of mesoglea (Fig. 4).

The frenular folds do not interrupt the circular muscle sheet; rather, the muscle fibers turn 90 degrees and run onto each face of the frenulum, extending radially instead of circularly (Fig. 4).

At the narrow frenular terminus, near the level of the rhopalial niche, muscle fibers do one of two things. Some of the fibers turn 90 degrees and run back into the circular muscle sheet of the upper velarium/subumbrella. Other fibers maintain a radial orientation, but splay out and terminate in the subumbrellar tissue in a fan-like manner (Fig. 4B).

High-magnification examination of actin-IH preparations verifies that the velarial muscle sheets are continuous with the frenular musculature, making the 90 degree turns at both the velarial and subumbrella attachments. In addition, muscle fibers are striated in all areas of the velarium and frenulum.

The velarium does not contain a separate layer of radial, smooth muscle like the velum of hydromedusae, so deformation of the velarium observed during turning (Gladfelter, 1973) presumably involves asymmetric contractions of the striated swim musculature.

Perradial muscle bands

The raised band of smooth muscle at each perradius originates just below the nerve ring (Fig. 5), runs to the top of the subumbrella, and extends into the quadrangular manubrium that hangs from the roof of the bell cavity (Fig. 6). Each perradial muscle bundle is comprised of nonstriated muscle fibers that splay out at its terminus below the nerve ring (Fig. 5D).

About one-third of the way up from the margin, the perradial muscle band gives off numerous outcropping fiber bundles that turn 90 degrees (into a circular direction) and extend a short distance across the subumbrellar muscle sheet (Fig. 5B, C). A lack of striations in actin-IH preparations, at high magnification, suggests that the muscle type is smooth. The smooth muscle fibers that run circularly interdigitate with the striated swim musculature, so at high magnification, the smooth nature of these fibers can be directly contrasted with the striated swim muscle (not shown). Despite our confidence in this, and additional supporting evidence, the smooth nature of the perradial fibers must be confirmed at the electron microscope level.

The four perradial muscle bands run into the quadrangular manubrium and form muscular ridges that continue in a radial direction into the elongated lips of the manubrium (Fig. 6). The bands maintain their integrity through the manubrial stalk but become less well defined in the manubrial body, where they give off a continuous sheet of smooth muscle cells that turn 90 degrees and connect with similar fibers from the adjacent perradial band (Fig. 6C, D). This organization continues into the four manubrial lips.

Musculature of the pedalia and tentacles

Actin-IH preparations revealed bands of longitudinal muscle throughout the length of the tentacles, from the pedalial attachment to the tip (Fig. 7). Presumably, circular muscle also exists, but it was not visible in our preparations. The muscle terminates at the tentacle-pedalium attachment through most of the cross section of the tentacle.

The blade-like pedalia also contain longitudinal muscle, but only on the oral side (Fig. 7A, B). The muscle fibers follow the curve of each pedalium, curling aborally and terminating abruptly no more than halfway across the width of the pedalium. The longest fibers are closest to the oral margin of the pedalium and extend to the tentacle attachment. The aboral-most fibers are short and extend a short distance along the length of the pedalium. In sectioned preparations, the muscle is found in a narrow layer at the base of the ectoderm. We were unable to see striations in either whole-mount immunohistochemical preparations or sectioned material; however, the arrangement of the muscle layers is such that determination of muscle type requires electron microscopical verification.

At the pedalium-subumbrellar attachment, the pedalial muscle fibers either attach to or are continuous with narrow bands of muscle that run into the subumbrella, turning in a circular direction. These bands run, at most, halfway to the perradius and terminate blindly in the subumbrellar tissue without fanning out (Fig. 7C). In contrast to the surrounding striated swim muscle, these fibers were judged to be of the smooth type.

Muscle of the rhopalial stalk

The rhopalial stalk contains muscle fibers that stain in actin-IH preparations. A narrow band of actin-IH-positive fibers runs in the oral wall of the stalk only, and splays out to terminate just above the attachment of the stalk to the subumbrella. We were not able to determine whether additional, non-staining muscle fibers existed in the aboral and lateral walls of the stalk, nor were we able to determine whether the stained fibers were striated or smooth.

Discussion

The subumbrella of the cubomedusa Tripedalia cystophora is lined with a nearly continuous layer of circular, striated muscle that provides the motive force for fluid ejection during a swim contraction (see Fig. 1 for a summary of muscle organization). The circular muscle sheet is continuous across each interradius, but is interrupted by a strip of radial, smooth muscle at each perradius. This perradial interruption separates the subumbrellar swim musculature into four quadrants that do not correspond to the four "flat sides" of the medusa. A broad, flat gastric pouch underlies each "flat side." The muscle sheet is attached to the exumbrella at the interradii, at the apex of the bell, at the junction of the subumbrella and the velarium, and around the rhopalial niche. However, the motor nerve net that innervates the swim musculature crosses the interradii and perradii (Coates and Satterlie, in prep.). The other interruption of the circular muscle sheet is at the nerve ring from which neurons of the motor nerve net emerge.

The circular muscle of the subumbrella is continuous with that of the velarium, suggesting that the velarium serves the same function as the velum of hydromedusae. As carefully noted by Conant (1898), Berger (1900), Hyman (1940), and Gladfelter (1973), the velarium is a derivative of marginal lappets and therefore an extension of the subumbrella. The important difference here is the lack of radial muscle in the velarium of cubomedusae. The radial muscle of the hydromedusan velum allows direct control of the shape of the nozzle during fluid ejection, and therefore direct control of turning. Similarly, cubomedusae turn by altering the shape of the velarium (Gladfelter, 1973), but the mechanism of muscular control is unknown, although the structure of the velarial frenula may provide a clue.

Each frenulum is a triangular bracket that reinforces the velarium at each perradius. The arrangement of muscle fibers on the frenula suggests that they are not passive buttresses. Muscle fibers of the velarial circular muscle sheet extend into each face of the frenulum and turn 90 degrees--in a radial direction. Near the site of the frenular insertion onto the subumbrella, below the level of the nerve ring, some of these fibers turn again and run back into the circular muscle sheet of the subumbrella. Other fibers splay out and terminate at the level of the rhopalial niche, maintaining their radial orientation. This suggests that, at the least, these radially oriented fibers provide an active reinforcement of the velarium during a swim contraction. However, they may do more. Examination of the swim motor nerve net of T. cystophora indicates significant differences in neuronal density above (subumbrella proper) and below (velarium) the nerve ring. The latter are more dense (Coates and Satterlie, in prep.). Within the velarium, nerve net fibers run into the frenula without interruption, suggesting that the frenula contract along with the velarium during a swim contraction. Interestingly, the motor nerve net has a noticeably higher density in the frenula than in the rest of the velarium.

So, what is the mechanism of directional nozzle formation by the velarium during turning? In hydromedusae, radial muscle is found throughout the exumbrella of the velum, providing an easy solution to this problem. In cubomedusae, however, directional nozzle formation requires asymmetrical enhancement of swim muscle contraction in the velarium. Two mechanisms immediately come to mind. Contractions of the subumbrellar swim musculature exhibit frequency-dependent facilitation, and rapid double pulses in the motor nerve net have been shown to produce large contractions (Satterlie, 1979). Recent observations suggest that these double-pulse contractions may be asymmetrical. If facilitatory events are related to nerve net density, the velarial frenula, with their radially oriented muscle fibers, could play a key role in directional nozzle formation and turning. Unfortunately, existing kinematic data (Gladfelter, 1973) do not focus on the frenula to see whether enhanced velarial contractions that produce directional nozzles are centered on these structures.

The other possibility involves dual innervation of swim musculature by a second nerve net, similar to that found in scyphomedusae (Romanes, 1876, 1877; Horridge, 1956a, b; Passano, 1965, 1973) and labeled with antibodies to RF-amide peptides (Grimmelikhuijzen et al., 2002). FMRF-amide-immunoreactive neuronal structures have been found in cubomedusae, but they are largely restricted to the rhopalia, nerve ring, tentacles, and manubrium (Coates and Satterlie, in prep.). In the subumbrella, immunoreactive fibers run in a tract along the perradial smooth muscle bands, but otherwise they are nearly absent in the circular muscle sheets.

The perradial smooth muscle bands of T. cystophora participate in two types of radial responses that pull the margin, or a portion of it, up and inward toward the center of the bell (Larsen, 1976). Protective "crumpling" involves contraction of all four sets of tentacles, curling of the tentacle pedalia inward into the bell opening, and an inward curling of the subumbrellar margin, in response to potentially injurious stimuli. This same type of behavior occurs asymmetrically during feeding, to bring a single tentacle into the bell for transfer of prey from the tentacle to the lips of the manubrium. It is likely that the perradial bands of smooth muscle participate in both of these behaviors. This raises a question about the circular extensions of this bundle found about a third of the way up from the margin. The fibers clearly are part of the radial band, but they turn 90 degrees to run in a circular direction partway across the swim muscle sheet. Contraction of these fibers along with the radial fibers could produce a wrinkle zone, or hinge, that would aid in pulling the tentacles into the bell during protective and feeding behaviors.

The function of the longitudinal muscle of the tentacles is obvious. Less obvious is the existence of circular muscle to oppose the action of the longitudinal muscle. Circular muscle did not show up with our staining techniques, but the brightness of the longitudinal muscle and the higher background in the tentacle tissue may have obscured it presence. Sections of fixed tissue suggest that circular muscle exists in the tentacles, particularly in the elevated rings of tissue that bear the batteries of nematocysts (pers. obs.).

The musculature of the tentacle pedalia is longitudinal and concentrated on the oral side. This presumably drives the inward contractions that bring the tentacles into the bell during feeding and protective behaviors. The only curious muscle structures associated with the pedalia or tentacles are the smooth muscle bands that radiate circularly from the points of attachment between the pedalia and the subumbrella. These narrow bands extend in both directions and terminate about halfway to the perradii on both sides of the pedalial attachment. It is possible that these fiber bundles also produce an active wrinkle zone to aid the inward bending of the pedalium.

Little is known about the musculature in the rhopalial stalk, and our preparations were not brightly stained. However, our data suggest that the muscle is asymmetrically arranged--found either exclusively, or in higher density, on the oral side of the stalk. Histological sections support this conclusion. From a functional point of view, this muscle organization would curl the rhopalium inward, away from the opening of the rhopalial niche, and hide it behind the small loop of exumbrellar tissue (hood) that partially covers the opening. Musculature of the rhopalial stalk may serve a protective function.

Acknowledgments

We thank Melissa Marie Coates for supplying specimens of Tripedalia used in this study. RAS was supported by NSF grant IBN-9319927 and NIH NINDS grant NS-27951. KST was supported by a Biology Research Experiences for Undergraduates assistantship through the School of Life Sciences, Arizona State University. GCG held a research assistantship in Biology at University of North Carolina, Wilmington.

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RICHARD A. SATTERLIE (1,2,*), KARA SUE THOMAS (2), AND G. CLARK GRAY (1)

(1) Center for Marine Science, University of North Carolina Wilmington, 5600 Marvin K. Moss Lane, Wilmington, North Carolina 28409; and (2) School of Life Science, Arizona State University, P.O. Box 874501, Tempe, Arizona 85287-4501

Received 23 May 2005; accepted 4 August 2005.

* To whom correspondence should be addressed, at Center for Marine Science, University of North Carolina Wilmington, 5600 Marvin K. Moss Lane, Wilmington, NC 28409. E-mail: satterlier@uncw.edu
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Author:Satterlie, Richard A.; Thomas, Kara Sue; Gray, G. Clark
Publication:The Biological Bulletin
Geographic Code:1USA
Date:Oct 1, 2005
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