Organization of the ectodermal nervous structures in jellyfish: scyphomedusae.
The subumbrellar swim musculature of scyphozoan jellyfish presents an interesting problem for neuronal control due to several unique features. First, the ectodermal muscle, composed of striated muscle cells, is broad and thin such that it represents a two-dimensional sheet that lines all or part of the subumbrellar tissue. Second, for coordinated muscle activation the neural activating system has to be capable of initiating coordinated contractions of this two dimensional muscle sheet from a variety of initiation sites located around the margin of the bell. The motor system thus has to be diffuse in organization and non-polarized in conduction properties, unusual traits in metazoan locomotory control.
The scyphozoan solution involves the use of such a diffuse, non-polarized nerve net for distribution of motor commands (Mayer, 1910; Bozler, 1926a, b; Horridge, 1956a). This is frequently cited as a primitive form of nervous system organization, and the phylogenetic position and body plan of cnidarians certainly invite this kind of speculation. However, the consistent organization of this type of motor control system in a wide variety of extant species argues that it also represents an efficient means of achieving the unique problem of non-polarized control of a broad two-dimensional sheet of effectors, and thus is more a consequence of the radial symmetry and a need to sense the environment from multiple sites spatially arranged around the margin of the bell. It is interesting that the hydrozoan jellyfish have found a very different mechanism for dealing with non-polarized conduction for activation of the two-dimensional effector sheet. They use gap junctions and electrical conduction within the muscle sheets, although in some species nerve nets are still used to augment the activation and coordination of effectors (see Satterlie, 2002).
For scyphomedusae, sensory information has to be conducted in a similar non-polarized manner since receptors are distributed around the margin of the bell, either via marginal tentacles or other marginal structures. Some of this information is conducted to the rhopalia, which themselves are primary sensory structures (Romanes, 1876, 1878; Passano, 1965). The rhopalia also contain the pacemakers that control the swim muscle contractions (Horridge, 1959).
This dual set of diffuse, non-polarized conducting systems in scyphomedusae was noted in behavioral experiments (Eimer, 1874, 1877; Romanes, 1876, 1878; Mayer, 1910; Bozler, 1926a, b), and later demonstrated with electrophy siological recordings by Horridge (1956a, b) and Passano (1965). A network of large neurons was identified as the motor system for coordination of swim contractions (called the Giant Fiber Nerve Net [GFNN]; Horridge, 1954, 1956a, b). The second nerve net (called the Diffuse Nerve Net [DNN]) was found to also innervate the swim musculature, either directly producing contractions, although of lesser strength, or having a modulatory role, enhancing ongoing swim contractions without directly producing them (Romanes, 1878; Mayer, 1910, Bozler, 1926a; Horridge, 1954, 1956a, b; Passano, 1965). The DNN also altered rhopalial pacemaker activity.
Recent morphological evidence supports the presence of two separate nerve nets in the subumbrella of scyphomedusae (e.g., Anderson and Schwab, 1981; Anderson et al., 1992; Carlberg et al., 1995). In a more general sense, various forms of tubulin antibodies have been used to stain neurons in cnidarians (Groger and Schmid, 2001; Galliot et al., 2009; Watanabe et al., 2009; Nakanishi et al., 2009, 2010), as have RFamide antibodies (Grimmelikhuijzen et al., 1996, 2002). Here, we use tubulin and FMRFamide antibodies to re-investigate the ectodermal nerve networks of ephyra and adults of Aurelia aurita, and of adults of several other species, as a test of the hypothesis that the tubulin antibody stains the GFNN and the FMRFamide antibody stains the DNN in the subumbrellar swim muscle sheets of scyphomedusae. Examination of other tissues seeks support for the additional suggestion that tubulin antibodies primarily stain motor networks in these animals (Nakanishi et al., 2009, 2010) and the FMRFamide antibody stains networks that include sensory components. An evaluation of this double staining has allowed more detailed description of some interesting aspects of neuronal architecture in scyphomedusae with regard to both networks. Finally, this study will form a basis of comparison for a similar examination of ectodermal networks in cubomedusae to highlight both common and unique features between these two medusoid groups.
Materials and Methods
The primary animal used in this work was Aurelia aurita Linnaeus, 1758, which was raised from a permanent polyp culture at the Center for Marine Science, University of North Carolina Wilmington, in a closed, recirculating system of kreisel aquaria. Polyps were fed daily with newly hatched Artemia nauplii and maintained at 31-33 ppt and 23 [degrees]C. Strobilation was induced by chilling the system for 3 weeks to 15[degrees]C, via an immersion chiller, and then allowing the system to return to 23[degrees]C over the course of a week. Ephyrae were collected in chambers by an overflow system and fed as for the polyps. As the medusae grew, they were placed in a succession of kreisel aquaria and maintained on the same daily feeding schedule. Adult medusae of up to 12-cm bell diameter have been raised in our system.
Adult specimens of Cyanea capillata (Linneaus, 1758) and Phacellophora camtschatica (Brandt, 1835) were collected from the breakwater at Friday Harbor Laboratories (University of Washington), Friday Harbor, Washington. Chrysaora quinquecirrha (Desor, 1848) adults were obtained from a permanent culture at the North Carolina Aquarium at Fort Fisher. Cassiopea xamachana Bigelow, 1892 was obtained from Carolina Biological Supply.
Prior to dissection, ephyrae and adults were relaxed in a 1:2 mixture of isotonic Mg[Cl.sub.2] (0.33 mol [l.sup.-1]) and seawater. The preparations were rinsed with seawater once quickly prior to immersion in fixative to minimize precipitate formation. Entire ephyrae or excised tissues of adults were placed in 4% paraformaldehyde in 0.1 mol [l.sup.-1] phosphate buffer (pH 7.4). Tissue pieces were fixed for 4-12 h with no difference in staining intensity. Fixed material was washed at least four times in 0.1 mol L1 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 (50%, 70%, 90%, 100%, 100%, 90%, 70%, 50%) to further permeabilize the samples. After blocking in 5% goat serum in phosphate buffer for 2-4 h, the tissue was incubated for 24-48 h in a 1:200 dilution of primary antibody (anti-[alpha]-tubulin or anti-FMRFamide, from the Developmental Studies Hybridoma Bank and Millipore Corp., respectively), washed in phosphate buffer (minimum of four changes), and incubated for 12-24 h in fluorescent-tagged secondary antibody (goat anti-rabbit for FMRFamide antibody and goat anti-mouse for ([alpha]-tubulin] antibody; tagged with AlexaFluor 488 or AlexaFluor 568 from Molecular Probes or FITC or TRITC from Sigma). For double stains, both primary antibodies were added together, as were secondary antibodies since the two primary antibodies had different host species (rabbit for FMRFamide and mouse for tubulin). The variation in incubation times produced no differences in staining for either antibody, so individual runs were adjusted within the tested limits for daily schedule convenience.
After another wash series in phosphate buffer, tissue pieces were cleared and mounted in a 9:1 mixture of glycerol and 50 mmol [l.sup.-1] Tris buffer (pH 9). Preparations were viewed in a Nikon epifluorescence microscope with FITC or TRITC filter cubes, and with an Olympus Fluoview 1000 confocal microscope.
Nineteen Aurelia ephyrae were used, eight each for the two primary antibodies and three for double stains. Tissue pieces were dissected from small and large adult Aurelia, including pieces from the margin (including tentacles), from the more peripheral subumbrella and more central subumbrella, the exumbrella, and the manubrium. Pieces of oral arms were not investigated. Similar dissections were made of the other medusae with the following number of specimens: Cyanea--9; Phacellophora--13; Chrysaora--7; Cassiopea--12.
The muscle arrangement for Aurelia ephyra and adults has been verified using phalloidin staining (Zimmerman and Satterlie, unpubl.).
Compressed z-stack images of [alpha]-tubulin- and FMRF-amide-labeled nerve nets from the swim musculature were printed onto 9 X 11-in paper, and 100 X 100-[micro]m quadrants were drawn using a stencil on each image. The areas chosen for evaluation were randomized within the circular muscle sheet. The angular orientation of each neurite was found relative to the axis of the bell margin designated as 180[degrees], and where 90[degrees] would indicate a radial orientation. A small protractor was used to measure the angle of displacement of individual neurites from the axis of the bell margin. A line was superimposed onto particularly sinuous neurites to determine the overall vector of displacement from the bell margin. Angle measures were sorted into eighteen 10[degrees] bins, spanning the range of 0 to 180[degrees]. Data were analyzed using JMP 7.0.7 (SAS Institute, Cary, NC). A chi-square test was utilized to test for significant differences in neurite orientation (number of neurites per bin) between the [alpha]-tubulin and FMRFamide-IR networks. P < 0.05 was considered significant for this test.
The localization of FMRFamide and tubulin immunoreactivity was best followed through a developmental sequence starting with the just-budded ephyra of Aurelia since the two stained subumbrellar networks showed very different relationships with the locomotory musculature in the early medusoid forms.
A tubulin-IR network was associated with the swim musculature of ephyra, staying within the limits of the circular muscle disc and the two radial muscle bands that run on either side of each ephyral arm. In the circular disc, the neurites showed a preferred orientation that followed the circular arrangement of the muscle (Fig. 1a). When passing into the radial muscle of the arms, the neurites changed their primary orientation and again followed that of the muscle.
In contrast, the FMRF-IR network was found throughout the arms and well beyond the limits of the radial muscle strips (Fig. 1b). It passed through the region of circular muscle with a more radial orientation (perpendicular to that of the tubulin-IR network), and continued across the subumbrella, extending into the manubrium, maintaining a radial primary orientation (Fig. 1c). In the lappets of the arms, away from the rhopalium, the FMRF-IR network was more random in neurite orientation relative to the strict radial orientation of tubulin-IR neurites (Fig. 2).
Both networks ran into the rhopalia and connected with rhopalial neuronal concentrations. Our data on rhopalial immunoreactivity to both antibodies were in agreement with the more detailed analysis by Nakanishi et al. (2009) and will not be described here.
Growth of ephyra included a disappearance of the arms and an extension of the circular muscle disc toward the manubrium. The tubulin-IR network followed this development of the circular muscle layer, but the neurites took a more random orientation to form a more morphologically nonpolarized nerve net structure. The FMRF-IR network continued to extend throughout the entire subumbrella and manubrium (Fig. 2b). Immunoreactivity was found throughout the lappets of the arms until they were no longer noticeable due to growth of the marginal tissue. The FMRF-IR network continued to extend to the bell margin after the tissue filled in to the mature discoid shape.
Organization of subumbrellar networks in adult medusae
The tubulin-IR and FMRF-IR networks showed overlapping staining in the subumbrella of all species of adult medusa, and stained different populations of neurons, as shown in double-label staining (Fig. 3). In Aurelia, the tubulin-IR network consisted of stout neurites up to 2.1 ptm in diameter (mean = 1.8 [+ or -] 0.07 SEM, n = 269 measurements from 6 specimens). Somata were not obvious in the densely stained network. In the FMRF-IR network, neurites were smaller, up to 1.6 [micro]m in diameter (mean = 1.5 [+ or -] 0.06 SEM, n = 354 measurements from 8 specimens). Somata were observed as distinct 3-[micro]m swellings.
FMRFamide-lR network. Dense networks were observed throughout the subumbrella of all species (Fig. 4), extending from the margin into (and throughout) the manubrium and the tentacles. Within the subumbrella, neurites appeared to be randomly oriented. However, a radial tendency was noted, particularly in Aurelia. To determine if the qualitative assessment was accurate, the orientation of neurites within the FMRFamide-IR networks of Aurelia was examined. Chi-square analysis results suggested that the distribution of neurite angle measures among bins was significantly different between the [alpha]-tubulin and FMRFamide-IR networks ([chi square](17, n = 491) = 122, P < 0.0001). Neurites in the FMRFamide-IR network were oriented more perpendicular to the axis of the bell margin (designated as 180[degrees]), while neurite orientation in the a-tubulin-IR network was independent of this axis (Fig. 5). The tubulin-IR neurites conformed to the classic diffuse nerve net arrangement.
Tubulin-IR network. Within the areas of the subumbrella that contain swim musculature, neurites of the tubulin-IR networks were evenly distributed but randomly oriented relative to the axis of the bell margin in all species (Figs. 5, 6a, b). Two exceptions are notable. First, in the region adjacent to a rhopalium, the neurites had a preferred orientation leading from the rhopalium (Fig. 6c). The second was found around the margin of the bell in Aurelia. Here, a narrow strip of tissue was devoid of swim musculature along the subumbrellar margin. While tubulin immunoreactivity extended across this marginal band to the margin and into the tentacles, there was a change in the structure of the network at the junction of the muscular region and the marginal band lacking circular muscle. The random, diffuse nature of the network in the muscular region condensed to form a dense accumulation of fibers at the junction, giving the appearance of a pseudo-nerve ring (Fig. 7). Stained neurons were found in the more peripheral region, but neurite orientation appeared more radial than random (Fig. 7). These neurons connected with accumulations of neurons at the bases of the marginal tentacles.
Marginal tentacles of Aurelia
Both antibodies stained neurons in the marginal tentacles, and in both cases, accumulation of cell somata was found at the tentacle bases (Fig. 7). FMRF-IR fibers continued into the tentacles at the base of the ectodermal epithelium. Throughout the tentacles, the network included stained epithelial cells that extended to the free surface (Fig. 8). The stained cells were distinct from nematocytes, which were unstained.
In the tentacle bases, tubulin-IR fibers extended into the tentacles as tract-like concentrations that gave rise to more diffuse ectodermal networks that were found throughout the tentacles (Fig. 7b, c). Stained epithelial cells, similar to those found with FMRFamide staining, were not present as components of the tubulin-IR tentacular network.
Rhopalia and rhopalial niches
Tubulin-IR staining of the rhopalia was hard to evaluate since the entire rhopalium tended to label. However, a dense array of stained fibers ran from the rhopalial base and diverged in the subumbrella, forming the tubulin-IR nerve net of the subumbrella (Fig. 6c).
FMRF-IR staining was more specific and included networks of neurons containing numerous stained epithelial cells (Fig. 9). The epithelial cells of the rhopalial body and stalk were consistent with suspected sensory cells described by Nakanishi et al. (2009). This is best seen in Cassiopea, where a high density of stained epithelial cells was present (Fig. 9b). Stained fibers of the rhopalial stalk were continuous with a dense network of fibers in the rhopalial niche. The niche network also included putative sensory cells. Similar staining was observed in the rhopalia and rhopalial niches of all species investigated, including the connections between neural elements of the rhopalia and networks in the niches. All areas included the presence of putative sensory cells.
Both FMRF-IR and tubulin-IR networks were found in the exumbrella of ephyra and medusae, although tubulin staining was less intense. Both networks were less dense (neurite density) than those found in the subumbrella for each respective antibody.
In the ephyra, exumbrellar nematocytes were mostly scattered rather than clumped (Fig. 10a, b). As a result, it was difficult to determine if the tubulin-IR or FMRF-IR network was associated with them. The FMRF-IR network was particularly well developed in the arms and lappets and showed the closest relationship with nematocytes.
In mature medusae, double staining revealed that the two networks were distinct and occasionally ran in parallel (Fig. 10c, d). At the margin, particularly in the region near the rhopalia, the FMRF-IR network was extremely dense and well stained. This included the exumbrellar portion of the rhopalial niche. The tubulin-IR network exhibited similar increases in density in the exumbrellar niche, although staining was less vibrant, and background staining made neurites difficult to distinguish. Both neuron types navigated the outer rim of the niche to merge with subumbrellar networks.
In adults, nematocytes were found in clusters, and both networks appeared to have some association with these nematocyst batteries (Fig. lOc-f). The nematocysts themselves stained brightly with the tubulin antibody (Fig. 10c). Neurites of the FMRF-IR network appeared to pass beneath nematocyst batteries and associate closely with nematocytes. In addition, there were 2 to 8 intensely stained nonnematocyte cells in each nematocyst battery (Fig. 1 Of). Occasionally these cells made contact with neurites of the FMRF-IR exumbrellar nerve net, but this was not always the case. Parallel fluorescent and DIC brightfield confocal images confirmed that FMRF-IR cells were closely associated with nematocytes (Fig. 10e, f). Immunoreactive cells and nematocysts were comparably sized at roughly 3-5 [micro]m across, although the latter frequently approached 6 [micro]m across.
The distinct and consistent staining of different neuronal populations with tubulin and FMRFamide antibodies suggests a tie to the two primary subumbrellar nerve nets described from behavioral and physiological studies of scyphomedusae (Romanes, 1876, 1878; Horridge, 1956a; Passano, 1965). In addition, other tissues, including the rhopalia, marginal tentacles, and exumbrella, exhibited similar staining of two distinct networks.
It might be expected that the tubulin antibody would label neurons of the FMRFamide-IR networks, and some doublelabeling was noted. However, the tubulin labeling of the FMRFamide-IR neurons was so dim it could only be noted at high magnification and with extremely bright illumination. In these double-label preparations, the tubulin immunoreactivity in the FMRFamide immunoreactive neurons faded within seconds, leaving only the FMRFamide labeling, which was both bright and robust.
In the same preparations, the tubulin labeling of neurons of the other network was extremely bright and did not show similar fading. Thus, despite this initial appearance of double-labeling, the staining of the two networks with FMRF-amide and tubulin antibodies was functionally distinct, and therefore useful in describing the double-innervation of the various medusan tissues.
The organization of the two subumbrellar networks, in adults and in ephyra-to-adult developmental sequences, supports the idea that the tubulin-IR network is the Giant Fiber Nerve Net originally described by Horridge (1954, 1956a), and also called the Motor Nerve Net (Schwab and Anderson, 1980; Anderson and Schwab, 1981). Similarly, the FMRF-IR network is found in all expected regions that correspond to the distribution of the Diffuse Nerve Net (Horridge, 1956a; Passano, 1965; Anderson et al., 1992), and includes morphological evidence suggestive of a sensory function. These correlations come with the necessary caution concerning the specificity of the antibodies, as there may be sub-networks within the stained systems. Furthermore, the FMRFamide antibody likely stained only a subset of RFamidergic cells and networks, as several distinct RFamides have been described in cnidarian tissues (Grimmelikhuijzen et al., 1996, 2002).
Giant Fiber Nerve Net
In the subumbrella, tubulin staining was restricted to the regions of swim muscle, including both circular discs and radial bands in the ephyra of Aurelia. As the ephyral arms "filled in" to produce juvenile medusae, the tubulin network maintained a distinct relationship with the circular musculature. The relationship was not strict, as both juvenile and adult Aurelia had a marginal ring of tissue devoid of circular muscle that still showed a tubulin-IR network. At the junction of the circular muscle disc and the non-muscular margin, the network was extremely dense, giving the appearance of a pseudo-nerve ring. The fibers that extended beyond this ring were continuous with stained fibers that ran into the marginal tentacles, so it is possible these neurons were part of a conduction system supplying the marginal tentacles. If so, the pseudo-nerve ring could represent an area of interaction between the subumbrellar and tentacular motor systems.
Additional verification of the link between the tubulin-IR network and the GFNN could come from double labeling with tubulin and taurine antibodies (Carlberg et al, 1995).
However, despite attempts to get taurine antibodies to work on several scyphozoan species and with multiple technique modifications, convincing taurine-IR staining was not obtained in our hands. However, intracellular recordings from subumbrellar neurons in Cyanea (Anderson and Schwab, 1983) provide direct evidence that the larger neurons (those that stain with tubulin) are components of the Motor Nerve Net. Analysis of neurite orientations within the tubulin-IR nerve net further supported the original descriptions of this network--that it is composed of large, randomly oriented neurons that form a nerve net distributed throughout the subumbrellar muscle sheet.
Diffuse Nerve Net
The Diffuse Nerve Net of scyphomedusae has been shown to serve a sensory function, but also to either provide a second source of motor activation of the swim musculature or to modulate ongoing contractions (Horridge, 1956a; Passano, 1965; Anderson et al., 1992). Neurites in this network are diffuse in organization, but with a significant radial orientation. The FMRFamide-IR nerve net of the subumbrella, tentacles, rhopalia, and exumbrella fits the distribution of this system as described previously (Romanes, 1878; Mayer, 1910; Bozler, 1926a; Horridge, 1954, 1956a, b; Passano, 1965). In the original descriptions, activation of the DNN involved a wave of marginal tentacle contraction, enhancement of swim muscle contractions, and alteration of the swim pacemaker output of the rhopalia. Furthermore, an exumbrellar component was suggested by Passano (1988, 2004) since transmission of activity in the DNN survived cuts through the margin of a medusa that blocked all but an exumbrellar route for information passage. In this way, the DNN may include sensory-motor pathways from most parts of the medusa.
The motor-sensory division of the two parallel pathways of the subumbrella may also be present in the tentacles. Within the tentacle bases, tubulin-IR fibers ran in condensed tracts that spread into tentacular networks that appeared to be deep to the epithelium and associated with the tentacular musculature. A separate FMRF-IR network was found in each marginal tentacle, again with densification in the tentacular base, as noted by Anderson et al. (1992). Within the tentacles, the FMRF-IR network appeared more superficial than the tubulin network and included stained epithelial cells, an appearance suggestive of sensory cells in cnidarians (Saripalli and Westfall, 1996).
Rhopalia and rhopalial niches
Similar putative sensory cells are found in the FMRF-IR networks of the rhopalial niches and on the rhopalial stalks. This is consistent with the more detailed analysis of rhopalial sensory cells of Nakanishi et al. (2009). In addition to the rhopalial sensory cells, putative sensory cells are also associated with dense FMRF-IR networks in the epithelium of the rhopalial niches of all species investigated. This raises an interesting question concerning the righting responses of scyphozoans. Scyphozoans, unlike several hydrozoan medusae, do not have true statocysts. The terminal end of each rhopalium contains a statolith, but it is covered with a tightly adhering epithelium. In view of the abundance of sensory cells on the rhopalium itself, it has been suggested that body position relative to gravity is detected on a larger scale, with the statolith acting as a "weight" only, as has been proposed for cubomedusae (Garm et al., 2011). The rhopalial sensory structures, specifically the concentrations of receptors known as the "touch plates" of scyphomedusae (Spangenberg et al., 1996), could participate as a sensory component of a larger scale statocyst-like function (Hundgen and Biela, 1982; Spangenberg et al., 1996; Arai, 1997). We can add the FMRF-IR network of the rhopalial niche as a possible component of this system because sensory cells are found in positions of possible contact with the rhopalium when the medusa is tilted. Contact of the weighted rhopalium with the sensory-rich niche tissues could thus be involved in detection of body tilt, in combination with rhopalial sensory structures. Additionally, sensory cells of the rhopalial stalk could act as stretch receptors stimulated by sag of the weighted rhopalium. In preliminary observations of anesthetized specimens of adult Aurelia, the statoliths did not produce enough rhopalial sag to contact the surrounding tissue of the rhopalial niche. However, contact with the niche hood and underlying lappets appeared to occur in unanesthetized individuals with each swim contraction. This suggests that phasic contact during swimming could contribute to gravity-detection in scyphomedusae.
The exumbrellar neuronal assemblages of scyphomedusae include separate tubulin-IR and FMRF-IR networks. A similar FMRF-IR exumbrellar network was described by Anderson et al. (1992). At first glance, the two networks appear to be associated with nematocytes, which are scattered in Aurelia ephyrae but form clusters in adults. Closer examination shows a more distinct association between nematocyst batteries and the FMRF-IR network, similar to that found in tentacles by Anderson et al. (2004). This includes stained epithelial cells within the batteries. Similar stained epithelial cells could not be detected in the tubulin preparations because all of the nematocytes stained brightly with the tubulin antibody.
The FMRF-IR cells of the nematocyst batteries are intriguing since they may represent sensory cells similar to those found in tentacles, rhopalia, and rhopalial niches (see also Anderson et al, 2004). Alternately, they could simply be immature nematocytes. Further investigation, including immuno-electron microscopy, should answer this question about the nature of these cells.
The FMRF-IR exumbrellar network could be a component of the DNN in scyphomedusae (Passano, 1988, 2004). Furthermore, just as it can have a modulatory influence on the subumbrellar swimming musculature (Romanes, 1878; Mayer, 1910; Bozler, 1926a; Horridge, 1954, 1956a, 1956b; Passano, 1965), it could have a similar modulatory influence on the discharge of exumbrellar nematocysts (see Anderson and Bouchard, 2009).
Summary and conclusions
The nervous system of scyphomedusae, as with most cnidarians, is frequently cited as a nerve-net-based system. While the nerve nets are the most conspicuous components of the scyphozoan nervous system, it is much more complex. The GFNN (MNN) represents the motor distribution system for activation of swim musculature. As such, it has to be capable of activating a broad, two-dimensional muscle sheet that follows the radial symmetry of the medusa. In addition, it must be able to activate this sheet from a number of initiation sites around the bell margin. The nonpolarized nerve net organization represents an efficient means of satisfying these activation parameters. The organization of the tubulin-IR network of the subumbrella best fits with the morphological and physiological properties of the GFNN.
Similarly, there is evidence for several functions of the DNN of scyphomedusae, including conduction of sensory information through the subumbrella and manubrium, and specifically to the rhopalia; modification of the swim rhythm; and conduction of DNN information to the swim musculature to modulate muscle activity (Romanes, 1878; Mayer, 1910; Bozler, 1926a; Horridge, 1954, 1956a, b; Passano, 1965, 1988, 2004). This latter property requires the same diffuse conduction as seen in the GFNN, giving the scyphomedusae two parallel nerve nets in the subumbrellar ectoderm, but with a distinct sensory component or sensory inputs. The FMRFamide-IR network best fits this organization. FMRFamide immunoreactivity in the subumbrella of scyphomedusae is contrasted by that of cubomedusae, in which the peripheral modulation of swim musculature apparently has been lost in favor of a more centralized FMRFamide-IR system (Eichinger and Satterlie, 2014).
The data presented here allow speculation concerning the function of this dual innervation of scyphozoan tissues. In all cases, the association with putative sensory structures in FMRFamide-IR networks and the absence of similar structures in the tubulin-IR networks suggests a consistent functional separation: sensory/modulatory/motor for the FMRFamide-IR systems and motor for the tubulin-IR systems. This appears to apply to the subumbrella, tentacles, rhopalia and rhopalial niches, and exumbrella.
Primary components of the scyphomedusan nervous system are the rhopalia, which contain complex neuronal pathways, some of which show a bilateral organization (Nakanishi et al, 2009). This ganglion-like nature of the rhopalia is combined with specialized sensory structures, much as the cerebral ganglia of higher invertebrates are associated with specialized sensory organs (Bullock and Horridge, 1965). The contrast then becomes one of body symmetry rather than of primitive traits. Attempts to draw a linear evolutionary origin from cnidarians to the bilateria will always be complicated by the differences in body symmetry and the resultant need for nervous system specialization related to those different body forms. The standard "textbook" suggestion that the nerve net is a primitive nervous system format that is later condensed into the ganglia and nerve cords of higher invertebrates is an oversimplification that ignores the specialized functions of the nerve nets and the existing neuronal condensation seen in the rhopalia of scyphomedusae.
Abbreviations: DNN, Diffuse Nerve Net; GFNN, Giant Fiber Nerve Net; MNN, Motor Nerve Net.
We thank the Director and staff of Friday Harbor Laboratories for their support, and the staff of the North Carolina Aquarium at Fort Fisher for the generous donation of Aurelia polyps and Chrysaora medusae. This work was supported by NSF grant IOS-0920825 and the Frank Hawkins Kenan Endowment (to RAS), and by a grant from Sigma Xi to JE. The monoclonal tubulin antibody was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biology, Iowa City, IA 52242.
Received 19 March 2013; accepted 4 February 2014.
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RICHARD A. SATTERLIE * AND JUSTIN M. EICHINGER
Department of Biology and Marine Biology and Center for Marine Science, University of North Carolina Wilmington, Wilmington, North Carolina 28409
* To whom correspondence should be addressed. E-mail: satterlier@ uncw.edu
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|Author:||Satterlie, Richard A.; Eichinger, Justin M.|
|Publication:||The Biological Bulletin|
|Date:||Feb 1, 2014|
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