Madreporite function and fluid volume relationships in sea urchins.
Sea urchins, like many other echinoderms, possess a conspicuous sieve-like opening to the exterior, the mad-reporite. This structure might be expected to have a function in fluid volume regulation, but no such role has ever been demonstrated. The madreporite is attached to a ciliated "stone canal" that connects through a series of passages to the animal's fluid-filled appendages, the tube feet. Together, these structures constitute the water vascular system, an important distinguishing feature of the phylum.
Observers often assume that seawater is drawn in through the madreporite by the stone canal cilia to hydraulically extend the tube feet. If, however, the connection to the madreporite is removed, the tube feet still continue to function for a long time; I have observed tube feet on broken pieces of sea urchin tests to remain distended and active for days. Further, the fluid within the water vascular system (ambulacral fluid, or AF) of echinoderms is not exactly identical in composition to seawater (Robertson, 1949; Binyon, 1964, 1966, 1976; Prusch, 1977; Ferguson, 1987).
Sea urchins have not been studied adequately, but precise osmotic measurements have been made on the fluid compartments of starfish (asteroids). Ambulacral fluid from starfish is sufficiently hyperosmotic to ambient seawater (about 6 mosmol [kg.sup.-1]) that some water would be drawn into this compartment from the medium, as well as from the perivisceral coelomic fluid (PCF) which is variably 1.5 mosmol [kg.sup.-1] more concentrated than seawater (Ferguson, 1990a). But in addition to this osmotic uptake, studies with fluorescent microbeads demonstrate that seawater does flow into the madreporite pores of asteroids (Ferguson, 1990b) and, to a lesser extent, of ophiuroids (Ferguson, 1995), and that it is distributed to peripheral parts of the water vascular system. When soluble dextran tracer was used to measure the flux of seawater into a starfish (Echinaster graminicola) through the madreporite, the rate was found to be about 5.5% of the body weight per day (Ferguson, 1989). However, this study also revealed that much of this inflow was diverted from the water vascular system to the perivisceral coelom, thus maintaining the fluid volume of the flexible body. (The Tiedemann's bodies that bulge from the ring canal of asteroids into the perivisceral coelomic space were probably the main route of the diversion (Ferguson, 1990b).) Further investigation showed that some starfish, such as the intertidal Pisaster ochraceus with its nearly impermeable integument, appear to rely heavily on transmadreporitic entry of seawater to maintain fluid volume (Ferguson, 1992), whereas others, such as the soft-bodied Pycnopodia helianthoides, may be proportionally more dependent on osmotic entry of seawater through a thin, permeable body wall (Ferguson 1990a, 1994). A balance between physical uptake of seawater through the madreporite and osmotic uptake through the integument appears to be typical for asteroids.
Sea urchins (regular echinoids), like asteroids, also have prominent madreporites, but their bodies are rigid. This latter feature would seem, at first, to obviate the need for most adjustments of body fluid volume or for any special mechanism (such as the madreporite) to take up seawater. On the other hand, the flexible peristome may allow for more variation in body fluid volume than has been appreciated, creating a potential need for fluid uptake. Or, the body fluid osmotic relationships of sea urchins may not be the same as those described in starfish (Ferguson, 1990a, 1992), and not adequate to maintain fluid homeostasis. So there is much uncertainty, and unfortunately literature bearing on these issues is desultory and often conflicting.
The most concerted previous attempt to evaluate the role of the madreporite in sea urchins was that of Fechter (1965; see review of Nichols, 1966), who glued small capillary tubes to the madreporites of five urchins (Echinus esculentus) so that, by observing the movement of tiny air bubbles in the tubes, he could measure any influx or efflux of fluid through them. Though his apparatus was reported as sensitive to a change of 0.8 [[micro]liter], he could detect no net movement of fluid over 24 h. Nevertheless, strong mechanical or chemical stimulation would cause all of the tube feet to contract simultaneously for a prolonged period, and a "slow" fluid outflow totaling 4 to 5 [[micro]liter] was seen. During such episodes, the PCF hydrostatic pressure rose 15-18 mm water (about 150-180 Pa) and remained elevated for about 10 h. Through these and other experiments, as well as on theoretical grounds, Fechter concluded that the primary role of the madreporite is to maintain an even pressure balance between the AF and ambient seawater, so that when the tube feet contract en masse, the displacement of their associated ampullae into the perivisceral coelom would not impart too much pressure on the peristome. Although a limited volume of fluid can be released through the madreporite, as Fechter (1965) suggested, prolonged contraction of all the tube feet is not natural; normally (except for momentary responses) when some tube feet contract, others are extending. Fechter's (1965) failure to see any seawater entry into the madreporite is important, but should be verified.
A more detailed study of the internal fluid pressures of Strongylocentrotus purpuratus and Lytechinus variegatus was carried out by Ellers and Telford (1992). They inserted a hypodermic needle attached to a pressure transducer through the peristome and recorded the pressure fluctuations associated with simultaneous contractions of the tube feet, but these were brief and only about 4% of the magnitude reported by Fechter (1965). Periodic peristomial movements (induced by the lantern) produced a fluctuating pressure change within the perivisceral coelom of about the same magnitude; the internal pressure tended to be negative (to -8.2 Pa) 70% of the time. Although not mentioned by them, this important finding points to another probable mechanism of PCF homeostasis - pressure filtration of water into the perivisceral coelom from higher pressure areas, particularly from the gut and the tube feet ampullae. Their study led Ellers and Telford (1992) to question Fechter's argument that the madreporite is primarily involved in acute pressure equalization.
Fechter's contention is, furthermore, not consistent with the anatomy of the madreporite and its associated stone canal. The form of the madreporite does not suggest a simple "relief valve." In sea urchins the madreporite typically consists of 300-400 pore canals partially filled with cilia that tend to forcefully exclude particles (Tamori et al., 1996). The inside diameter of each pore is about 21 [[micro]molar], which is variable because the pore can contract to less than half its resting size in response to acetylcholine (Takahashi and Tamori, 1988; Takahashi et al., 1991; Tamori et al., 1996). Thus, the total cross-sectional space of the madreporite openings is only about 0.12 [mm.sup.2]. The normal fluid exchanges through such a restricted opening are likely to be too small to affect the fluid volume of the animal except, perhaps, over the long term. Nevertheless, fairly large fluid volume changes (milliliters) can take place in sea urchins within hours in response to osmotic challenges (Lange, 1964; Stickle and Ahokas, 1974). Logic suggests that this last adjustment must involve an initial displacement of the peristome, followed by rapid water and ion movements across the gut and other permeable surfaces. Moreover, in preliminary work leading to the present investigation, sea urchins (Strongylocentrotus pallidus and S. purpuratus) with obstructed madreporites were repeatedly able to return to a near-normal body weight (and volume) within hours after removal of 1-3 ml of their PCF, clearly showing that the madreporite is not needed for acute large-scale volume changes.
Thus, the functions of the prominent madreporite system of sea urchins remain unknown, and the normal osmotic differences that might exist between their various body fluids and the media have not been accurately measured. In this study methods previously applied to asteroids are used to examine two questions. First, does seawater enter the sea urchin madreporite, and if it does, is the quantity sufficient either to affect the function of the tube feet or to stabilize body fluid volume? Second, are osmotic differences between the internal fluids of sea urchins and the outside medium consistent, and could they contribute to inflation of the tube feet or to augmentation of general body fluid?
Materials and Methods
Treatment of specimens
Work was conducted on four congeneric species of sea urchins collected from waters around Friday Harbor, Washington: Strongylocentrotus purpuratus, S. pallidus, S. droebachiensis, and S. franciscanus. They were kept in shallow tanks with flowing seawater and fed ad libitum with kelp picked up along the shore. In some cases, the madreporites of "test" animals were obstructed by first scraping the structure with a needle and blotting up the soft tissue, and then sealing the area with freshly mixed, finely ground hydraulic cement. After the cement had hardened for about 10 min, the animals were returned to seawater. These plugs were tolerated very well, and they showed no sign of failure over the course of any of the experiments.
Fluorescent microbead experiments
Influxes of seawater through the madreporite and into the water vascular system were demonstrated as follows. Two small specimens of S. droebachiensis (3.1 and 3.4 g) were selected and placed for 5 days in a dish of aerated seawater (250 ml) to which was added 1 ml of a suspension of 0.2 [[micro]molar] YG "Fluoresbrite" carboxylate beads (Polysciences, Inc.). Then, after they were rinsed in seawater, the animals were cut into several parts to facilitate further handling. These were fixed overnight in 10% formalin and decalcified under refrigeration for 10 days in several changes of 5% EDTA and 10% formalin. After rinsing in tap water, pieces were selected and trimmed to suitable size for sectioning (3-5 mm diameter). Attention was focused on pieces containing the madreporite complex, the aboral half of the stone canal, and representative parts of the aboral and oral body walls with tube feet and ampullae attached. These were frozen onto stubs with Tissue-Tek OTC compound, sectioned in a cryostat to 20-50 [[micro]meter], picked up on glass slides, mounted with cover slips over glycerine jelly, and examined under epifluorescence using a Nikon system. Only animals with intact madreporites were used for these observations; obstructing the madreporite would have destroyed the major area to be studied and would have precluded sectioning. Further, previous studies on asteroids and ophiuroids (Ferguson, 1990b, 1995) suggested that particles could enter the water vascular passages only with bulk flow of seawater through the madreporite pores.
Soluble fluorescent tracer experiments
An effort was made to quantify the influx of seawater through the madreporite with the soluble, high molecular weight tracer, fluorescein isothiocyanate dextran (FID), as was previously done with asteroids (Ferguson, 1989, 1994). To accomplish this, 100 mg FID (73,100) (Sigma Chemical) was dissolved in dishes of gently bubbled seawater (800 ml) containing pairs (one "test" and one unaltered "control") of smallish S. pallidus (81-155 g). At 24-h intervals, samples of 0.5 ml PCF were taken from the sea urchins through a 26-gauge hypodermic needle inserted through the peristome. Unlike asteroids, an adequate AF sample could not be obtained from the small lamellar ampullae of these animals, and larger sea urchins (including S. franciscanus) require so much medium that work with them is impractical. For analysis of the PCF, the collected fluid was centrifuged, and 0.1-ml aliquots of the supernatant were vortexed in tubes with 3.5 ml phosphate buffer. The fluorescence of the samples in these tubes was read in a Turner fluorimeter equipped with 2A and 47B primary filters and a 2A-12 secondary filter. Measurements were compared with samples of medium that had been similarly processed, as well as samples of medium that were diluted 1:10. This method can detect FID in the PCF to concentrations about 0.25% that of the medium.
Long-term effects of madreporite obstruction
Two sets of experiments tested the effect of long-term obstruction of the madreporite on behavior, tube feet activity, and maintenance of body fluid volume. It was assumed that small, gradual adjustments in the volume of individual sea urchins would be observable as systematic variations in their wet body weights, and be due mainly to the level of inflation of the tube feet, or to adjustments in the position of the peristome. Matched test and control groups of S. droebachiensis were selected and observed for several weeks. In one case the animals were fed, and in the other they were not. By using a standardized weighing procedure in which animals were allowed to pre-drain for 2 min on paper towels, daily weight measurements accurate to about 0.1 g were obtained. From a series of such measurements on an individual, a least squares linear regression yielded an average daily change, which was expressed as the percentage of net daily variation from the mean body weight over the period. This deviation was referred to as the "change in body weight index" or [Delta]WI.
At the conclusion of the experiments, a more specific measurement of the water content of the body parts was made as follows: First, a specimen was inverted for 15 min over a beaker, and any fluid released from its anus was collected. An incision into the body cavity was then made through the equator of the body and the PCF drained out and collected. The animal was then dissected onto tared weighing trays, with the body wall and lantern, the gonads, and the remaining gut separated out. Each collection of tissues was weighed and then dried to constancy (about 24 h) at 75 [degrees] C, and reweighed. Body water index (BWI) was calculated as the percentage of the wet body weight of the intact animal represented by the drained PCF plus the difference between the total wet and dry weights of parts other than the gut. Gut water index (GWI) similarly was taken as the percentage of the intact animal's wet weight represented by the weight of the collected anal water plus the gut water (difference between wet and dry weights of gut tissue); the water content of any food or feces within the gut was included. The osmotic concentrations of the collected PCF of the animals were also monitored.
Osmotic difference between body fluids and ambient seawater
Finally, osmotic comparisons were made between the body fluids of separate groups of sea urchins and their ambient seawater. For the PCF, about 0.5 ml of fluid was removed from the body cavity with a 26-gauge sytinge needle inserted diagonally through the anus, and was immediately placed in a capped microcentrifuge tube. After flushing the syringe with seawater, an equivalent "reference" sample of ambient seawater was taken and placed in a similar tube. Both samples were then centrifuged (5 min) and 10-[[micro]liter] aliquots were then analyzed in a vapor pressure osmometer (Wescor 5500). To compensate for any drift in the instrument, the reference seawater sample was analyzed first with three successive replicates on the sample, which remained sealed in the instrument chamber. The PCF was analyzed next in the same way, followed by another set of analyses on the reference seawater. The mean of the six reference seawater measurements was then subtracted from the mean of the three PCF measurements to yield the osmotic difference. Specimens of S. francis-canus were sufficiently large that individual tube feet could be seized with a hemostat, cut off, and drained into a centrifuge tube, and their ambulacral fluid (AF) analyzed in the same way. Since these procedures were designed to measure small osmotic differences between body fluids and ambient seawater (a few mosmol [kg.sup.-1]), the precision of the method was determined by analyzing in the same manner 12 replicates of separate seawater samples substituted for the body fluids. These had a standard deviation of [+ or -]2.24 mosmol [kg.sup.-1].
The effects of obstructing the madreporite on [Delta]WI, BWI, and GWI of test and control animals were evaluated with the Mann-Whitney U test, using the z statistic to estimate significance. A correction for multiple tests for the three procedures may be made using the Bonferroni procedure, in which the selected confidence limit for a single test is divided by the number of tests. In that case, the 95% confidence limit would be set as P [less than] 0.017. In the osmotic studies, the mean differences between body fluids and ambient seawater were evaluated with a Student's t test.
Uptake of fluorescent microbeads
As was observed in previous studies on asteroids and ophiuroids (Ferguson, 1990b, 1995), microbeads were taken up extensively into the exposed epidermis of the two urchins examined. Substantial numbers were also found within the water vascular system, especially in the madreporite and stone canal [ILLUSTRATION FOR FIGURES 1-12 OMITTED]. Figure 13 provides a diagrammatic orientation to the madreporitc region of the body. In the madreporite, some beads were seen in the lumen of the outer passages of the fine pore canals, and some within the pore cells, most concentrated in their apical portion [ILLUSTRATION FOR FIGURES 1-3 OMITTED]. In the mid-region of the canals, numerous beads were found throughout the pore cells [ILLUSTRATION FOR FIGURES 3-5 OMITTED]. At the lower end of the pore canals, the abundance of beads retained diminished markedly as the canals extended towards the region of their confluence (i.e., the sub-madreporite ampulla) and the stone canal beyond [ILLUSTRATION FOR FIGURES 5, 6 OMITTED]. In the stone canal itself, a thick stringlike aggregate of beads and perhaps other material extended down the lumen [ILLUSTRATION FOR FIGURES 7, 8 OMITTED]. A few beads were also seen in the upper axial organ [ILLUSTRATION FOR FIGURE 7 OMITTED], in the lumen of the rectum (found attached to the madreporite pieces that were sectioned), and in the peritoneum (or coelomocytes) near the rectum [ILLUSTRATION FOR FIGURES 9, 10 OMITTED]. Some beads were found in the lumen of tube feet, especially in the lower (oral) region of the body [ILLUSTRATION FOR FIGURES 11, 12 OMITTED], but they were not plentiful. Many, perhaps most, tube foot sections failed to reveal any beads in the lumen.
Table I Levels of FID (percentage of medium concentration) in the PCF of madreporite obstructed (Test) and unaltered (Control) Strongylocentrotus pallidus after incubation in FID seawater Days in medium Weight (g) 1 2 3 4 5 Test 87.5 0 0 0 0 0 112.4 0 155.3 0 Control 80.7 0 0 0 0 0 94.7 0 116.1 0
Uptake of FID
In three attempts with test and control pairs of S. pallidus exposed to high levels of FID in the medium for 24 h (Table I), measurable buildup of the tracer could not be detected in the PCF. In one case, exposure was continued for an additional 4 days, but the substance still could not be observed in the body fluid. I therefore decided that further tests with the FID method were unwarranted.
Long-term effects of madreporite obstruction
A group of madreporite-obstructed specimens of S. droebachiensis, and a control group, were observed for 27 days while they were maintained in an aquarium with running seawater and kelp for food. No differences were noticed in the behavior of the two groups. Their tube feet remained active, and animals in both groups were observed to roam the aquarium and climb the sides, feed on the kelp, and rapidly right themselves if upset. No significant differences were measured in the variation of their daily weights ([Delta]WI) or, at the end of the experiment, in their body water content (BWI) or its osmotic level (Table II). However, the gut water index (GWI) of [TABULAR DATA FOR TABLE II OMITTED] [TABULAR DATA FOR TABLE III OMITTED] the "test" animals was found to be significantly (P [less than] 0.006) lower than that of the control group (Table II). When a similar experiment was carried out for 21 days on unfed animals, the gut water indexes remained low in both tests and controls, and not significantly different, but the test animals lost weight significantly more rapidly than the controls (P [less than] 0.013) (Table III). Otherwise, no differences were noticed in the behavior or well-being of the two groups.
Osmotic differences from ambient seawater
Precise osmotic measurements were made on the PCF of groups of three species (S. pallidus, S. droebachiensis, and S. franciscanus) and compared to ambient seawater (Table IV). All showed mean osmotic levels significantly above that of their ambient seawater (P [less than] 0.01), with a combined mean ([+ or -]SE) elevation of 2.66 [+ or -] 0.39 mosmol [kg.sup.-1]. Clean samples of ambulacral fluid were obtained from the large tube feet of S. franciscanus, and they had a mean ([+ or -]SE) osmotic level (7.94 [+ or -] 1.04 mosmol [kg.sup.-1]), significantly above that of the PCF for this species (P [less than] 0.01).
Table IV Osmotic difference between body fluids of sea urchins and their ambient seawater Species Fluid n mosmol [kg.sup.-1] [+ or -] SE S. pallidus PCF 12 +2.86 [+ or -] 0.69 S. droebachiensis PCF 12 +2.68 [+ or -] 0.77 S. franciscanus PCF 12 +2.44 [+ or -] 0.59 S. franciscanus AF 12 +7.94 [+ or -] 1.04 Seawater(*) 12 -0.54 [+ or -] 0.65 * Repetitive samples analyzed just like body fluids (all body fluids are significantly different from seawater (P [less than] 0.01)).
These experiments with fluorescent microbeads and soluble tracer (FID) show that in contrast to previous work in asteroids (Ferguson 1989, 1990b) entry of seawater into the madreporite of echinoids is very limited. The continued, apparently unimpaired activity of tube feet even after 27 days of madreporite occlusion further indicates that these structures are inflated mainly by some other means. Certainly their elevated osmotic AF (+7.94 mosmol [kg.sup.-1] in S. franciscanus) must make a contribution, although redistribution of fluids between body compartments may also be involved. Likewise, the failure to detect significant amounts of FID in the PCF shows that there can be but little transmadreporitic inflow of seawater to generate that fluid. If minor entry did occur, it was completely masked by the natural cleansing processes that must exist within the animals and the limits of sensitivity of the method. In a parallel study in which this method was used on the starfish Echinaster graminicola, uptake of FID into the PCF was easily measured (in spite of evident fluid cleansing) and found to be about 1.5 [[micro]liter] [g.sup.-1] [h.sup.-1], out of a total madreporite influx of 2.3 [[micro]liter] [g.sup.-1] [h.sup.-1] (Ferguson, 1989). The transmadreporite influx of seawater in starfish thus appears to be directed mainly at keeping the flexible body inflated, a process that is largely unnecessary in sea urchins. Moreover, sea urchins do not have Tiedemann's bodies, which may be asteroid specializations for passing fluid to the PCF. With their rigid test obviating much of the need for fluid volume control, sea urchins appear to rely mainly on a higher PCF osmotic level (+2.66 mosmol [kg.sup.-1] reported here) than seen in most asteroids (Ferguson, 1990a), and on a net negative hydrostatic pressure reportedly produced within the test by the movement of the lantern and peristome (Ellers and Telford, 1992).
Is there any consistent flow of seawater at all into the sea urchin madreporite? Recent work by Tamori et al. (1996) on isolated madreporite pieces indicates that the pores usually maintain an inward flow that is regulated by changes in their diameter. Cilia within the pores tended to eject particles aborally. The present work shows that a small proportion of minute particles (beads) suspended in the medium can still enter the pore canals of intact animals. These particles are extensively trapped, especially by middle pore cells [ILLUSTRATION FOR FIGURE 1-6 OMITTED]. Many of the particles that succeed in passing through the length of the pore canals become entangled in a string of material within the lumen of the stone canal [ILLUSTRATION FOR FIGURE 7, 8 OMITTED], which is lined with ciliated cells filled with granules (Rehkamper and Welsch, 1988). Nevertheless, a few particles pass by these obstacles and are transported to the lumen of distal tube feet, where they are finally phagocytized by coelomocytes [ILLUSTRATION FOR FIGURE 11, 12 OMITTED]. Particles and phagocytic debris also accumulate to some extent in the axial gland, near the rectum, which may be a site of phagocytic egress [ILLUSTRATION FOR FIGURE 7, 9, 10 OMITTED]. These observations lead to the conclusion that a very slow bulk flow of seawater passes through the madreporite and down the stone canal to the tube feet, and that the pore cells help to remove foreign material from this entering stream.
Although the FID study was not sensitive enough to show such a slow flow, other data support its existence. In working with the starfish Pisaster ochraceus, the transmadreporite seawater influx was estimated from the rate of weight loss in animals with an obstructed madreporite (Ferguson, 1992). That value was 2.2 [[micro]liter] [g.sup.-1] [h.sup.-1] (which was close to the rate determined from the FID studies on Echinaster (Ferguson, 1989)). Using a similar approach, madreporite seawater influx in S. droebachiensis can be roughly estimated by comparing the mean weight loss differences ([Delta]WI) in test and control animals (Table III). If all the difference between the two groups was due to inhibited madreporite influx, that influx would amount to about 0.3 [[micro]liter] [g.sup.-1] [h.sup.-1]; it is equal to a maximum total inflow of not more than 0.13 ml of seawater a day into the madreporite of a 200-g sea urchin. This rate is at least 70 times lower than the ones calculated for starfish. Although this approach is not very precise, the order of magnitude of the figure obtained seems consistent with differences seen in the microbead observations, negative FID experiments, and other observations, including the negative findings of Fechter (1965).
Does such a low rate of madreporite seawater entry have any functional significance? The long-term weight-change studies (Tables II and III) give some indication that it might, eventually. Although the differences resulting from madreporite obstruction were small and not noticeable in the behavior of the animals, unfed test specimens gradually became unable to maintain body weight, whereas fed test specimens had a slightly higher gut water index. The reasons for these effects are unknown, but possibilities include a gradually increasing deficit of AF that may have limited the number of tube feet that could be used at any one time; or a reduced PCF volume that might have hampered the animal's ability to protrude its lantern apparatus. These kinds of effects could be related. The water vascular system is connected indirectly to the perivisceral coelom through an opening between the upper end of the stone canal and the adjacent axial sinus [ILLUSTRATION FOR FIGURE 13 OMITTED] (cf., Jangoux and Schaltin, 1977; Bachmann and Goldschmid, 1978). So deficiencies in fluid content in one compartment might affect the functions of the other, given enough time. Thus, this limited fluid uptake appears to "top off" a fluid balance that is largely met by the osmotic differences between the body fluids and the surrounding seawater, and by net negative periodic hydrostatic pressures in the perivisceral coelom.
The madreporite may have other functions that are unrelated to fluid volume regulation. These include chemosensing, a passage for entry of pheromonal stimulants, and excretory roles. None of these has as yet been convincingly demonstrated.
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|Author:||Ferguson, John C.|
|Publication:||The Biological Bulletin|
|Date:||Dec 1, 1996|
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