Hydrobiologia 216/217: 45-49, 1991.R. B. Williams, P. F. S. Cornelius, R. G. Hughes & E. A. Robson (eds), 45Coelenterate Biology: Recent Research on Cnidaria and Ctenophora.© 1991 Kluwer Academic Publishers. Printed in Belgium.

Rhopalium development in Aurelia aurita ephyrae

Dorothy B. SpangenbergDepartment of Pathology, Eastern Virginia Medical School, Norfolk, Virginia 23501, USA

Key words: jellyfish, ephyra, rhopalium, kinocilia, mechanoreceptor, ultrastructure

Abstract

Rhopalia of developing ephyrae were examined using the SEM and TEM at 24 h intervals followingstrobilation induction. Kinocilia are shorter in the ephyra stage than in polyps. A few ephyra-type kinociliaare found in rhopalia as early as 24 h after induction, before a distinct rhopalium is seen. By 72 h, theshorter kinocilia predominate and are almost as numerous as in ephyrae (120 h). Many of the kinociliaare associated with mechanoreceptor cells (MR) found in the rhopalia. These MR cells are comparedto those reported for medusae. Although newly released ephyrae lack a touch plate, the MR cells in theirrhopalia along with the statocyst and neuromuscular system apparently enable these organisms to detectand respond to gravity.

Introduction

Medusan rhopalia have been associated with thepulsing, swimming and orienting of scypho-medusae for many years. Rhopalia have marginalganglia which control the animal's pulsation(Romanes, 1876). Rhopalia also act as gravi-ceptors which enable medusae to right themselvesafter tilting (see Passano (1982) for review) and toperceive tilting during swimming (Schwab, 1977).

Much less is known about rhopalium develop-ment and function in ephyrae. Statolith formationhas been described during ephyra developmentand during exposure to various experimentallyinduced conditions (see Spangenberg (1986) forreview). Yoshida & Yoshino (1980) followed thedevelopment of ocelli. Horridge (1956) reportedon neuron distribution in rhopalia of ephyrae andSchwab (1977) recorded marginal ganglion poten-tials. Complementing the work of Romanes(1876) with adult medusae, Spangenberg et al.

(1989) found that excision of rhopalia similarlyresulted in the ephyra's inability to pulse rhythmi-cally or pulse, even during parabolic flight.

Developmental and morphological studies ofephyral rhopalia were done to determine whetherthe rhopalia have all the functions of medusanrhopalia, and eventually to detect abnormal devel-opment of rhopalia of ephyrae under experimentalconditions. The Aurelia Metamorphosis Test Sys-tem (Spangenberg, 1984) was used in this studyto track the development of rhopalia under con-trolled laboratory conditions.

Material and methods

Jellyfish were collected in Norfolk, Virginia,U.S.A. and cultured in artificial seawater(Spangenberg, 1965) for at least five years. Theseorganisms are Aurelia aurita (auct.) of an un-named variety. (They do not fit previous descrip-

46

tions of east coast A. aurita varieties given byAgassiz (1862) or Kramp (1942).) Polyps wereinduced to strobilate at 28 C using 10-5 Miodine (Spangenberg, 1967). The distal segment ofstrobilae and the 'heads' of polyps were severedfrom the organisms and prepared for scanningelectron microscope (SEM) studies (Spangenberg& Kuenning, 1976). All of the rhopalia of 5ephyrae were examined at 24 h intervals up to120 h. Micrographs, including stereopairs, wereused to examine kinocilia length and morphology.Rhopalia of newly released ephyrae were exam-ined in the SEM and transmission electron micro-scope (TEM) (Spangenberg, 1976).

Results

Rhopalium development

Using the SEM, it was observed that during stro-bilation, rhopalia develop non-synchronously atthe bases of the perradial and interradial ten-tacles, before the distal portion of the tentaclesdisappears. The most highly developed rhopaliumis described for ephyrae at each 24 h period. Con-siderable rearrangement of tissues takes place atthe site where rhopalia will form. Within 24 h,broadening of the bases of the perradial and inter-radial tentacles begins and increases up to 48 h.Rhopalia form as the distal portions of the ten-tacles disintegrate.

Prior to strobilation induction, numerous kino-cilia are found at the bases of the perradial andinterradial tentacles (Fig. 1A). The kinocilia(Fig. 1B) are 17-22 pm long, have bulbous tipsand are surrounded by a circlet of microvilli. Alsoon the tentacles are cnidocils of nematocysts,which are straighter than kinocilia and each hasa mound of tightly associated microvilli at itsbase. In the ephyrae and polyps of Aurelia, twotypes of cnidocil are found which correspond tomicrobasic heterotrichous eurytele cnidocils(4 pm or less) and atrichous isorhiza cnidocils(8-10 pm). Few cnidocils are found on develop-ing rhopalia. On the surface of the polyptentacles filamentous 'worm-shaped' structures(1-4 x 0.5 pm) are also seen which could perhaps

be microvilli or dried mucus (as suggested byBlanquet & Wetzel (1975) for Chrysaora sur-faces).

After strobilation begins at 24 and 48 h, a fewshorter kinocilia of the ephyra type, 8-12 pmlong, are seen in the pre-rhopalium region at thetentacle base (Fig. C, 1D). Significant numbersare not found until 72 h when oval rhopalia format the bases of the perradial and interradial ten-tacles. These newly formed rhopalia (Fig. 1E) areca 100 ,tm long and 55 pim wide. Their predomi-nant cilia are these shorter kinocilia, 8-12 pmlong, surrounded at the base by a circlet of shortmicrovilli (Fig. 1F). A few longer kinocilia resem-bling those found on polyp tentacles can also befound. The surface of the rhopalium has numer-ous tiny round separate globules of various sizes(which may be secretory granules) and many tinymicrovilli. Only a few of the 'worm-shaped' fila-mentous structures are now found.

After 96 h, distal ephyral segments resembleephyrae. Their rhopalia are thinner (35-50, m)and have an hour-glass shape. No tentaclesremain and some of the ephyrae have begun puls-ing weakly.

Within 120 h, ephyrae from the most distal seg-ments are free-swimming. On the oral side of theephyra, the rhopalia (Fig. 1G) have numerousshort kinocilia (Fig. H), especially on the proxi-mal two-thirds of the rhopalia where mechano-receptor (MR) cells are found. At the rhopalialtips, in contrast few kinocilia are found. Rhopaliaof newly developed free-swimming ephyrae are ca100 pm long and 30-40 pm wide. While they areusually seen lying flat when viewed from the oralside, they are capable of bending into commashapes, with the statocyst touching the area oflappet bifurcation. The rhopalial cilia, includingkinocilia, are motile (in contrast to cnidocils) andisolated rhopalia placed on a substratum moveslowly along using ciliary motion.

Rhopalial MR cells

Using the TEM two types of ciliated cell resemblethe types I and II MR cells of the touch-plate of

A B

/

POLYP POLYP-TYPE KINOCILIA

C D - -

/

POLYP-TYPE KINOCILIA,24 H STROBILIA EPHYRA-TYPE*

E F --I'l - o, ',

/ ... . . .. .

I ,

EPHYRA-TYPE KINOCILIA72 H STROBILIA NO POLYP-TYPE

G H

-.. . .......I z ~~I

,,Im,

120 H FREE-SWIMMING EPHYRA EPHYRA-TYPE KINOCILIA

Fig. 1. Kinocilia changes during rhopalium formation at 28 °C. Oral view ofjellyfish. h = hypostome; r = rhopalium; m = micro-villi; t = tentacle; 1 = lappet. A) Bases of tentacles where rhopalia will develop during strobilation (circle); bar = 100 gm. B) Typeof kinocilia found in circle of A; bar = 5 m. C) Site of rhopalium formation (circle) 24 h after strobilation induction;bar = 100 pm. D) Type of kinocilia found in circle of C; bar = 5 m. E) Early rhopalium formation 72 h after induction;bar = 100 pm. F) Type of kinocilia on rhopalium (circle) of E; bar = pm. G) Rhopalia of free-swimming ephyrae at 120 h after

induction; bar = 100 pm. H) Konocilia of cells of rhopalium (circle) of G; bar = 5 m.

4/A7

f-

. _ .

I-

48

medusae (Hundgen & Biela, 1982) and similarcells of higher organisms (Budelmann, 1988).These ephyral MR cells are elongate, oftenreaching from the surface to the mesoglea. Theyare found primarily in the proximal two-thirds ofthe rhopalia and run perpendicular to the axis ofthe mesoglea. One cell type has cytoplasmicwhorled bodies as in type I cells but neither celltype has myofilaments in the base as reported fortype II cells of medusae. Neurites which extendfrom both types of MR cell descend into a plexusof neurites near the mesoglea. An occasional non-polarized synapse is found between two neurites.

A third type of ciliated cell which resembles anMR cell is found adjacent to the statocyst. Thesecells are not elongated and have one or moreneurites which enter the neurite plexus.

Discussion

Rhopalium development has been depicted indeveloping ephyrae by Spangenberg & Kuenning(1976). In the present study, rhopalia were exam-ined daily in strobilae developing at 28 C, inwhich free-swimming ephyrae are formed within120 h. Since a distinct rhopalium is not founduntil 72 h after induction of strobilation, con-siderable growth and differentiation of rhopaliatake place between 72 and 120 h.

Examination of strobila segments before 72 h,using the SEM, revealed that polyp-type kinociliadecrease in number at the pre-rhopalium site, per-haps through degeneration of polyp cells. Evenbefore tentacle loss is complete, tiny statolithsform at the bases of the tentacles of strobilaewithin 58 h of strobilation induction (Spangen-berg, 1976). Since polyp cells do not make stato-liths, the presence of lithocytes with statolithsindicates that these cells have already differenti-ated.

After 72 h, finding significant numbers ofephyra-type kinocilia may signal the formation ofnew rhopalial cell types such as MR cells.Changes in surface morphology as the rhopaliadevelop, such as a decrease in number of fila-mentous 'worm-shaped' structures and an

increase in numbers of small globules, may also bedue to a change of cell types during rhopaliumformation.

In most ephyrae, tentacle loss is not completebefore 96 h when pulsation begins. The onsetof pulsing indicates that the neurons of the giantfiber nerve net and those of the rhopalia whichinteract with them have developed. Schwab(1977) reported that marginal ganglion primordiaare electrically quiet for some time after the ten-tacles disappear. Marginal ganglion potentials arerecorded when the ephyra begins to pulse.

Cilia of elongated receptor cells at the tentaclebases of Aurelia polyps (Chia et al., 1984) maycorrespond to the kinocilia found at polyp ten-tacle bases. These receptor cells, however, aremorphologically unlike the MR cells found inephyral rhopalia. Changes in kinocilia duringrhopalium formation probably represent, there-fore, the formation of new MR cells. One cell type,which has cytoplasmic whorled bodies, stronglyresembles the type I MR cell of medusae(Hundgen & Biela, 1982). These authors empha-size the resemblance of this cell type to hair cellsof the statocysts of the gastropods Limax andArion which also have whorled bodies. Otherciliated rhopalial cells which probably form anewduring strobilation are the non-elongated cellswith kinocilia located near the statocyst and alsosome of the epidermal cells.

Cnidocils like those on the surface of therhopalia of Aurelia were described by Blanquet &Wetzel (1975) in Chrysaora 'scyphopolyps'. Thesestructures are components of nematocysts whichmay either have developed during rhopalium for-mation, or have migrated from the tentacles intothe rhopalia (nematocytes are capable of migra-tion). Likewise, some of the polyp epidermal ten-tacle cells may be retained in the rhopalia sinceoccasionally a cell with a polyp-type cilium isfound.

To summarize, the development of ephyralrhopalia occurs within 120 h after the induction ofstrobilation at 28 C. Changes in ciliation occuron the surface of tentacle bases where rhopaliaform and on developing rhopalia. Cells withephyra-type short kinocilia appear as early as 24 h

49

after strobilation induction and are predominantin rhopalia by 72 h when most polyp-type ciliahave disappeared. Three new cell types withwhich surface kinocilia could be associated werefound. Hundgen & Biela (1982) stated that MRcells of the touch-plate together with the statocystcomprise the organ by which A. aurita senses thedirection of gravity. Although newly releasedephyrae lack an organized touch-plate, they havea statocyst and, throughout the rhopalium, atleast 3 different types of MR cells. These rhopalialcomponents are apparently sufficient for ephyraeto sense gravity and to evoke pulsing/swimmingresponses.

Acknowledgements

I thank Mr Jim Slusser and Ms Suzanne Davisfor preparing tissues for ultrastructure studies andMs Debbie Miller for the illustrations. Thisresearch is funded through grants # NAG 2-343and # NAG 2-508 from the National Aeronauticsand Space Administration, USA.

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