Cnidocyte-supporting cell complexes (CSCCs) discharge nematocysts into targets upon coincidental stimulation of specific chemoreceptors and contactsensitive mechanoreceptors. In addition, CSCCs in the tentacles of at least one species of sea anemone discharge nematocysts into targets vibrating at specific frequencies. In seawater alone, these CSCCs discharge nematocysts preferentially at 55, 50 and 75 Hz. In the presence of 10−7M N-acetylneuraminic acid (NANA) or mucin, the CSCCs discharge nematocysts preferentially at the lower frequencies of 0, 5, 15, 30 and 40 Hz. Furthermore, the stereocilium bundles (SBs) within ciliary cones of CSCCs elongate significantly from a mean length of 6.08 /on in seawater to 7.14 μm in 10−7M mucin. The responses of (1) shifting the optimal frequencies for discharging nematocysts to lower frequencies and (2) elongating the SBs both exhibit dose-dependency and temporal adaptation to chemosensitizer. We conclude that these responses are controlled by CSCC chemoreceptors for N-acetylated sugars. We suggest that specific size-classes of SBs respond to specific frequencies of vibration, since the dose-response parameters to NANA depicting the relative abundances of SB size classes measuring 3–4, 5 and 7 μm correlate with dose-response parameters for the discharge of nematocysts into targets vibrating at 75, 55, and 30 Hz. Treating tentacles with cytochalasin disorganizes the SBs of ciliary cones and decreases the number of frequency optima for nematocyst discharge without significantly affecting nematocyst discharge into static targets. Thus, ciliary cones on CSCCs are vibration-sensitive mechanoreceptors that can be tuned by chemoreceptors to specific, lower frequencies by the elongation of SBs.
Cnidocytes, the stinging cells diagnostic of Cnidaria, are among the most complex eukaryotic cells known (Novikoff and Holtzman, 1976). Each cnidocyte contains a nematocyst or some other type of cnida consisting of a capsule containing a highly folded and eversible tubule (Skaer and Picken, 1965). Cnidocytes discharge nematocysts in response to appropriate stimulation of contact-sensitive mechanoreceptors and specific chemoreceptors (Thor-ington and Hessinger, 19886). This discharge culminates in the rapid eversion of the tubules (Holstein and Tardent, 1984). Nematocysts are used to capture prey, to defend against predation (Ewer, 1947), and to inflict damage to other cnidarians in aggressive interactions (Francis, 1973). For some types of nematocysts, the everting tubules penetrate the target to inject potent toxins (Hessinger, 1988), while for others they adhere to the surface or entangle appendages of the target organism (Mariscal,1974).
At least two classes of chemoreceptors on supporting cells (Watson and Hessinger, 1987) detect substances derived from suitable target organisms and then predispose adjacent cnidocytes to discharge nematocysts in response to physical contact (Thorington and Hessinger, 1988,a). One class of receptors exhibits broad specificity for amino compounds and the other binds certain free and conjugated N-acetylated sugars (Thorington and Hessinger, 1988 a).
At least one species of sea anemone discharges nematocysts into vibrating targets. In the absence of proper chemical stimulation, cnidocytes in tentacles of the sea anemone, Haliplanella, discharge nematocysts preferentially into targets vibrating at 50–55 Hz, followed by 65–75 Hz, and 30 Hz (Watson and Hessinger, 1989a). Following chemosensitization, maximal discharge occurs at 5, 15, 30 and 40 Hz; frequencies that match the movements of the swimming prey (Watson and Hessinger, 1989a).
Ciliary cones cover the apical surface of cnidocyte-supporting cell complexes (CSCCs) in the tentacles of acontiate sea anemones (Mariscal et al. 1978; Pantin, 1942). Each cone consists of a single kinocilium surrounded by a circlet of short microvilli and several, more peripheral, circlets of stereocilia (Bigger, 1982; Westfall, 1965). The tips of the stereocilia converge so that the array forms a conical, stereocilium bundle (SB). Whereas the kinocilium and microvilli originate from the cnidocyte, the stereocilia and the chemoreceptors for N-acetylated sugars are located on two or more supporting cells that surround each cnidocyte (Watson and Hessinger, 1987). Thus, in anemone tentacles, the CSCC constitutes the functional and structural unit of nematocyst discharge (Watson and Hessinger, 1989b).
In this report, agonist-induced changes in the length of the SBs are described and analyzed in relation to the frequency-dependent discharge of nematocysts.
Materials and methods
Bovine submaxillary mucin (type I), JV-acetylneuramimc acid (type VI), cytochalasin B, and MgCl2.6H2O were obtained from Sigma Chemical Co., St Louis, MO; gelatin from MCB, Cincinnati, OH; Instant Ocean artificial sea salts from Aquarium Systems, Inc., Mentor, OH; and encysted embryos of Artemia salina from San Francisco Bay Brand, Newark, CA.
Maintenance of animals
Monoclonal specimens of the sea anemone, Haliplanella luciae, were reared in flat-bottomed, glass trays in Instant Ocean artificial seawater (ASW) at 32 %o and 23(±1)°C. The animals were maintained on a 12 h light:12h dark photoperiod and fed freshly hatched Artemia nauplii twice weekly. Experiments were performed approximately 72 h after feeding for the purpose of maximizing nematocyst discharge (Thorington and Hessinger, 1988 b).
Measurement of SBs of ciliary cones
Specimens were anesthetized for 60 min in magnesium/artificial seawater (Mg/ASW), consisting of equal parts of 0.6 M MgCl2 and ASW. The tentacles were excised and prepared as wet-mounts. The microscope focus was adjusted to bring the equatorial aspect of the tentacle tip into view. Ciliary cones near the tips of the tentacles were photographed in profile using phase-contrast optics at a final magnification of ×100 (Nikon Diaphot, 40DL LWD objective). The SBs consist of a truncated conical array of stereocilia surrounding a single cilium. In phase-contrast optics, the SB of each ciliary cone appeared as a triangular projection extending from the tentacle surface. For each agonist, the first 50 suitable SBs encountered by moving a × 15 scale loup along the periphery of the tentacle tip were measured directly from the negative to the nearest 1.0 μm. Suitable SBs were those appearing in focus along the total SB-length, and with clearly identifiable tips and bases (see Fig. 1). The tips of the SBs were ascertained by a sharply defined, abrupt termination of the stereocilium bundle from which the cilium projected. The bases of the SBs were identified by a decrease in contrast at the proximal-most 1–2 μm of SB length. SBs lacking this decrease in contrast were assumed to originate above or below the plane of focus and were not measured. This assumption was confirmed on live specimens by rocking the focus. Error caused by oblique orientations of the SBs was minimized by measuring SBs in focus along the total SB length. Although the experiments were not performed blind, observer bias was minimized by selecting the first 50 suitable SBs encountered.
Validation of methods for measuring SBs
To test whether measurements of SBs obtained using the ×40 objective lens adequately depicted the population of SBs on the tentacle, measurements of SBs under comparable conditions were performed using a ×40 objective and rounded to the nearest 1.0 pm and compared with those using a ×100 objective lens (DIC optics, Olympus BH-2S) rounded to the nearest 0.2 μm (Table 1). Since the mean lengths and standard deviations, and the percentage elongation observed for the experimental groups, were essentially comparable despite the higher accuracy of the × 100 objective, we concluded that the ×40 objective adequately described SB population dynamics. In addition, since multiple SBs could be captured per frame using the lower-power objective lens, we could more easily keep the total incubation interval constant in the agonist solutions. This was essential for comparing agonist effects on SB length with VSM frequency responsiveness. For these reasons, experimental measurements were performed using the ×40 objective.
Effects of N-acetylneuraminic acid (NANA) on individual SBs
Several tentacles and a small piece of the oral disc were excised together to restrict axial rotation of the tentacles in wetmount preparations. One region of a single tentacle was selected at random from the group and photographed in Mg/ASW. The tentacles were transferred to Mg/ASW containing 10 −7 M NANA and the same region of the tentacle was photographed through a series of optical sections. Photographic negatives from the experimental group were placed directly over those from Mg/ASW controls on a light box. Individual ciliary cones were identified by several topographical markers used conjointly, including: their locations along the length of the tentacle; their orientations with respect to the tentacle surface; and the locations and orientations of adjacent cones. The lengths of SBs were measured directly from the negatives to the nearest 0.5 /an using a Nikon S2B stereomicroscope at x20 magnification and fitted with an ocular measuring reticule calibrated from a stage micrometer.
Dose-responses of SB elongation to NANA
Animals were anesthetized in Mg/ASW and then transferred to NANA at specified concentrations ranging from 10−18 to 10∼SM in Mg/ASW. After 5 min in the NANA solution, tentacles were prepared as wet-mounts and photographed at × 100 magnification. At each concentration, 50 suitable SBs were measured to the nearest 1.0 μm using a X15 scale loup as described above. The abundance of specific size classes of SBs was plotted against the concentration of NANA. The dose of NANA that produced a half-maximal increase in SB length (LK0.5; L for length) was calculated for each dose-response from a Hanes-Woolf type plot (Dorgan and Hessinger, 1984).
Dose responses and frequency responses of nematocyst discharge
Animals were removed from the mass culture and placed in 35 mm diameter Falcon plastic dishes filled with ASW. They were allowed 3–4 h to recover normal responsiveness, at which point the ASW was replaced with NANA in ASW at specified concentrations ranging from 10−18 to 10−6M. After 10 min in the NANA solution, tentacles were touched with static test-probes (0 Hz) or with vibrating test-probes (5, 15, 30, 40, 55 and 75 Hz; Watson and Hessinger, 1989a).
Each test-probe consisted of a 2-cm segment of 0.14 mm diameter, nylon fishing line (Stren Brand, 2-lb test, DuPont) coated at the tip with 30% (w/v) gelatin to a thickness of approximately 200 /on (Watson and Hessinger, 19896). A bend in each probe ensured a snug fit into the glass capillary tubes (internal diameter=0.27 mm). The capillary tube was held in place by a custom-machined teflon fitting offset from the driveshaft of a galvanometer by 4.0 mm (Watson and Hessinger, 1989a). The galvanometer was driven by a function generator set to the sine-wave function so that the tip of the probe oscillated 140 pm as the galvanometer drive-shaft rotated through an arc of 2° (Watson and Hessinger, 1989a). Frequencies and displacements of probe vibrations were calibrated using a stroboscope. Vibrating probes have a linear frequency response from 5 to 100 Hz for displacements at the tip ranging from 35 to 700 μm (unpublished data).
After contacting the tentacles, the gelatin-coated tips of the probes were fixed in 2.5 % glutaraldehyde in ASW for 1 min and then stored in distilled water at 4 °C before being examined. Probes were prepared as wet-mounts and the microbasic p-mastigophore nematocysts discharged into the gelatin coating were counted in single fields of view at ×400 magnification (0.16 mm2) of an inverted microscope (Nikon Diaphot). On a given day, four replicate probes were used for each experimental condition or frequency, one probe for each of four anemones. The data are presented as the mean of two daily means ± the standard error. The concentration of NANA that produced half-maximal discharge of nematocysts CKo.s) was calculated (Dorgan and Hessinger, 1984). A typical frequency response consisted of 21 test-frequencies ranging from 0 to 100 Hz at 5-Hz intervals and, thus, of 168 values (21×4×2).
Anemones were anesthetized in Mg/ASW and then incubated in 10 pg ml-1 cytochalasin B in Mg/ASW for 30 min. Animals were placed in ASW for 30 min to permit recovery from the anesthetic before nematocyst discharge was tested for cytochalasin-treated and untreated animals, as follows: (1) dose response to NANA in ASW using static probes (0 Hz); and (2) frequency response using vibrating probes at 10−7M NANA in ASW, and in ASW alone.
Variation in the data for nematocyst counts
The coefficient of variation for replicate test-probes averaged about 33% of the mean value at a given frequency/condition. Variance component analysis indicated that there was no significant variability (P>0.5) between identical experiments performed on different days. Thus, although we present data for nematocyst counts as the mean of two daily means and calculate standard error on the basis of n=2, the standard error could have been calculated on the basis of n=8, since the day to day variability was not significant. The analyses as presented are conservative.
Unless otherwise specified below, the results of statistical analyses are summarized in the text or in figure legends as follows: the type of test used; and the relevant P-value.
Measurements of SB length
Data were subjected to one-way analysis of variance (ANOVA; Sokal and Rohlf, 1969) to test for heterogeneity between group means. Upon finding significant heterogeneity, multiple comparisons of the treatments versus a seawater control, were performed by the Dunnett i-test (Dunnett, 1964; Zar, 1974), which utilizes differing critical values for each comparison in order to compensate for the loss of independence. In all cases, significant differences are reported at a P value of 0.01 or less and non-significant differences at a P value of greater than 0.05.
Frequency responses of nematocyst discharge
Nematocyst counts obtained for the test-frequencies under different, experimental conditions were subjected to one-way ANOVA (Sokal and Rohlf, 1969) to test for heterogeneity among test-frequencies. Newman-Keuls tests (Sokal and Rohlf, 1969), which systematically compare mean values pairwise, were performed to identify significant frequency-response peaks within each treatment at a P-value of <0.05. The total data set was subjected to two-way ANOVA to test the effect of treatment by frequency interaction (after adjusting for main effects due to treatment and frequency). Treatments were then compared pairwise by two-way ANOVA.
Data for dose responses to NANA comparing the number of nematocysts discharged following cytochalasin treatment with the number in the absence of treatment were analyzed by the Fisher i-test for independent samples (Sokal and Rohlf, 1969). Spearman Rank correlation (Sokal and Rohlf, 1969) was used to correlate the dose of NANA with the abundance of specific size classes of SBs and with the discharge of nematocysts into test-probes vibrating at specific frequencies.
Scanning electron microscopy
Specimens were anesthetized in Mg/ASW, treated either in cytochalasin in Mg/ASW as described above, or in Mg/ASW alone, and then fixed in 2.5 % glutaraldehyde in ASW for 1 h. The tissue was rinsed in ASW, dehydrated in acetone, and criticalpoint dried. Specimens were mounted on stubs, sputter-coated with gold-palladium to a thickness of 20 nm, then observed at 25 kV using a Philips 515 Scanning Electron Microscope.
Mucin induces SBs of ciliary cones to elongate
Tentacle preparations had numerous ciliary cones at the epithelial surface with each SB forming a conspicuous, sheath-like array at the base of the cilium (Fig. 1).
The mean lengths of SBs before, during, and after treatment in 10−7M mucin were significantly heterogeneous between the groups. In Mg/ASW, the mean length of the SBs was 6.08 μm (Fig. 2A). Within 1 min of exposing tentacles to 10−7 M mucin, the SBs significantly elongated by 17.4% to a mean length of 7.14gm (Fig. 2A). This increase included a greater than threefold increase in the percentage of SBs measuring 7 μm in length, a comparable decrease in the percentage of SBs measuring 5 μm in length, and the appearance, for the first time, of SBs measuring 10 μm and 11 μm (Fig. 2B).
Mucin-induced elongation is reversible
Approximately 1 min after removing the mucin, the SBs shortened to a mean length of 6.98 μm (Fig. 2A), remaining significantly larger than seawater controls and indistinguishable from SBs in mucin (Fig. 2A). Beginning 5 min after removing the mucin and continuing through 30 min, the SBs were indistinguishable from seawater controls despite a progressive decrease in the mean length (Fig. 2A) caused by an increase in the proportion of SBs measuring 5 μm in length (Fig. 2B). This shortening of the SBs to control levels was half-completed within 2.5 min (Fig. 2A). Within 10 min after removing the mucin, SBs measuring 10 μm or longer were not detectable (Fig. 2B).
Individual SBs are affected differently by NANA
The effects of 10−7M NANA on the lengths of the most-abundant size-class of SBs in Mg/ASW (5.0 tan) were determined to the nearest 0.5 gm. Of the 50 SBs sampled, 88% elongated in the NANA solution, with the greatest elongation observed for three SBs increasing by 60 % to a final length of 8.0 μm (Fig. 3A). The mean increase was by 34.6% to a final length of 6.73 μm (Fig. 3A). Disregarding the SBs that did not elongate, the mean elongation was by 39.2 % to a final length of 6.97 μm (Fig-3A). No SBs were observed to shorten.
To determine the effects of NANA on other size classes of SBs, a total of 90 SBs in Mg/ASW were selected, 10 each at 0.5 μm increments of initial length ranging from 4.0 to 8.0 μm. Following 10 min exposure to 10∼7M NANA, the SBs either remained the same length (24 %) or increased in length by as much as 3.0 μm (Fig. 3B), with 19 % of the population elongating by 2.5 pm or more, and the average increase by 1.4 μm. In a relative context, the greatest elongation was observed for the shortest SBs, 4.0 pm, of which two increased by 63 % (Fig. 3B). Considering the entire range of initial SB lengths, the mean elongation was by 25 % to 33 %, depending on whether the non-responsive SBs were included in the estimate.
Adaptation, of NANA-induced elongation of SBs
The SBs measured before exposure to NANA, during brief exposure to NANA, during prolonged exposure to NANA, or after prolonged exposure to NANA followed by brief exposure to NANA at a higher dose, exhibited heterogeneity in mean length between the treatments (Fig. 4).
In Mg/ASW, the mean length of the SBs was 5.28 μm (Fig. 4A). After 5 min in 10−7 M NANA, the SBs elongated significantly to a mean length of 6.78 μm (Fig. 4B). After incubating the entire animal for 24h in 10−7M NANA, however, the SBs shortened to lengths indistinguishable from seawater controls with a mean length of 5.54 gm (Fig-4C). Following 24 h in 10−7M NANA and then 5 min in 5×10−5 M NANA, the SBs elongated to a mean length of 6.50 gm (Fig. 4D), significantly larger than seawater controls but indistinguishable from CSCCs exposed to 10−7M NANA for 10 min.
Adaptation of NAN A-sensitized discharge of nematocysts
The frequency-dependent discharge of nematocysts was tested for anemones incubated in ASW alone, during brief exposure to NANA, during prolonged exposure to NANA, and during prolonged exposure to NANA followed by brief exposure to NANA at a higher dose. One-way ANOVA confirmed the existence of ‘peaks’ in the frequencyresponse curves (Fig. 5).
Newman-Keuls tests, performed to identify statistically significant peaks within each treatment, indicated that for the naive controls, CSCCs discharged nematocysts preferentially into probes vibrating at 55, 50 and 75 Hz (Fig. 6A) with a minor peak also appearing reproducibly at 30 Hz (Fig. 5A; Watson and Hessinger, 1989a). After 10 min in lOM mucin (Watson and Hessinger, 1989a) or NANA, CSCCs discharged nematocysts preferentially at 30, 5 and 15 Hz, followed by 40, and 0 Hz (Fig. 6B). After 24 h in 10∼7 M NANA, however, the frequency optima for nematocyst discharge were similar to those of naive CSCCs (Fig. 5C), with significant optima occurring at 55 Hz, followed by 75, 50, 30 and 65 Hz (Fig. 6C). After 24 h in 10−7 M NANA followed by 10 min in 5 ×10−5 M NANA, the frequency optima shifted to 40 and 15 Hz, followed by 30 Hz (Fig. 6D), optima otherwise comparable to values for animals exposed to 10−7 M NANA for 10 min (Figs 5B,6B), except for fewer nematocysts discharging into probes at 5 and 0 Hz (Figs 5D,6D).
NANA-induced elongation of SBs is size class-specific and dose-dependent
Increasing doses of NANA caused SBs measuring 7 gm or larger to increase in abundance while causing SBs measuring 5 fan or smaller to decrease in abundance (Fig. 7). The SBs measuring 4 /an and smaller decreased from a maximum abundance of 36 % in Mg/ASW to 2 % in 10−7M NANA (Fig. 7A). The proportion of SBs measuring 5 /an decreased by more than threefold (Fig. 7B), while the proportion of SBs measuring 7 μm increased by fourfold (Fig. 7D), and the proportion of SBs measuring 8–10 μm increased by fivefold (Fig. 7E). The calculated doses of NANA that produced half-maximal changes in abundance for the different size classes of SBs (LK0.5 values) are provided in Table 2.
NANA-induced nematocyst discharge is frequencyspecific
The numbers of nematocysts discharged into test probes vibrating at 75 Hz and 55 Hz each decreased approximately twofold with increasing concentrations of NANA (Figs 8A,B), while the numbers of nematocysts discharged into test probes vibrating at 40, 30, 15, 5 and 0 Hz each increased approximately twofold (Figs 8C-G). The dose-response curves for vibrating probes appeared to be hyperbolic in shape (Figs 8A-F), whereas the dose-response curve for static probes (0 Hz) was biphasic in shape (Fig. 8G; Thorington and Hessinger, 1988 a; Watson and Hessinger, 1989b). The sensitivity of nematocyst discharge to NANA, expressed in Ko 6 values, decreased as the frequencies of the vibrating test probes were decreased from 40 to 0 Hz (Table 2), with K0 5 values at specified frequencies spanning more than six orders of magnitude of NANA concentrations.
SB length correlates to frequency-specificity of discharge
Parameters of dose responses to NANA for changes in abundance of specific size-classes of SB and for the discharge of nematocysts at specific frequencies were compared to determine if specific size-classes of SBs respond to specific frequencies. Two parameters were compared: (1) the similarity in shape of the dose-response curves by using Spearman-Rank analysis (Sokal and Rohlf, 1969); and (2) the similarity in K0.5 values by calculating a ratio of the respective K0.5 values (i.e., LK0.5/K0.5). The analysis comparing the shapes of the dose-response curves determined the extent to which NANA-induced changes in SB length are comparable to NANA-induced changes in nematocyst discharge. The ratio of calculated K0.5 values determined the extent to which these effects of NANA exhibit comparable sensitivities. A calculated ratio of 1.0 indicates a perfect correlation in the doses of NANA inducing half-maximal responses. These analyses compare dose responses irrespective of the scales used on the ordinate axis.
The dose-response parameters for NANA-induced changes in the abundance of SBs measuring 4 μm or smaller correlated to those for the discharge of nematocysts at 75 Hz with a highly significant, positive correlation in the shapes of the curves and a nearly perfect K0.5, ratio (Table 3). Dose-response parameters for NANA-induced changes in the abundance of SBs measuring approximately 5 μmin length correlated best with those for the discharge of nematocysts at 55 Hz with highly significant, positively correlated shapes and a reasonable K0.5 ratio (Table 3). The parameters for NANA-induced changes in the abundance of SBs measuring 7 /on in length correlated best with those for the discharge of nematocysts at 30 Hz. The shape of the dose-response curves exhibited a highly significant, positive correlation, and a good K0.5ratio (Table 3).
The analyses of data for SBs measuring 8 μm or larger were inconsistent, with the best, positive correlation in shape occurring with data for nematocyst discharge at 15 Hz, and the best K0.5 ratio occurring with the data for nematocyst discharge at 5 Hz (Table 3).
The data for the NANA-induced discharge of nematocysts into test-probes vibrating at 40 Hz correlated best with those for SBs measuring approximately 7 μm in length. However, the correlation was weaker than for discharge at 30 Hz with the dose-response curves exhibiting a less-significant correlation in shape, and a less ideal K0.5 ratio (Table 3).
The data for nematocyst discharge into static probes were inconsistent, with the best correlation in shape occurring with data for SBs measuring 7 μm in length, and the best K0.5ratio occurring with data for SBs measuring 8–10 μm (Table 3).
Cytochalasin B disorganizes SBs and alters frequency-specific discharge of nematocysts, but not discharge into static test-probes
Frequency-dependent nematocyst discharge over the range of 0 to 100 Hz exhibited highly significant heterogeneity between anemones treated in ASW alone, in 10 μgml-1 cytochalasin B for 30 min followed by 10 min in ASW, in 10−7M NANA for 10min, or in 10μgml-1 cytochalasin B for 30 min followed by 10−7M NANA for 10min (Figs 9,10).
In ASW alone, the CSCCs showed significant frequency optima for discharging nematocysts at 55, 50 and 75 Hz (Figs 5A,6A). For anemones treated in cytochalasin B and tested in ASW, a single significant peak of nematocyst discharge was detected at 80 Hz (Figs 9A,10A). In 10”‘M NANA, the CSCCs discharged nematocysts preferentially into test-probes at 30, 5 and 15 Hz, followed by 40 and 0 Hz (Figs 5B,6B). For anemones treated in cytochalasin B and tested in 10−7M NANA, the CSCCs discharged nematocysts preferentially only into test-probes at 0 Hz (Figs 9B,10B). Whereas cytochalasin treatment significantly decreased the number of, and altered the location of, frequency optima for discharging nematocysts into vibrating test-probes, cytochalasin treatment did not significantly affect the dose-dependent discharge of nematocysts into static test-probes, even at permissive levels of significance (Fig. 11). Cytochalasin treatment was observed to disorganize the bundle of stereocilia comprising the SBs of ciliary cones (Figs 12A,B), causing most of the SBs to ‘disappear’ from the surface of five tentacles (half-time=3.7 min; data not shown) leaving only sparse 3–4 μm SBs poorly contrasted by phase-contrast optics.
SBs elongate in response to N-acetylated sugars
Upon binding free or conjugated N-acetylated sugars, receptors on supporting cells predispose cnidocytes to discharge nematocysts in response to physical contact (Figs 8G,11B; Thorington and Hessinger, 1988a; Watson and Hessinger, 19896), and induce SBs to elongate in a dose-dependent manner (Fig. 7). Such elongation is completely reversed within 5 min of removing the chemosensitizer from the medium (Figs 2A,B); it has a half-time of about 2.5 min (Fig. 2A).
SBs elongate by varying extents
Despite comparable initial lengths, 5.0 μm SBs exposed to 10−7 M NANA elongated on average by about 35 % to a final length of 6.7 μm, although the observed increase ranged from 0 to 3 μm (Fig-3A). A similar pattern was observed for the other size classes of SBs (Fig. 3B). Thus, the SBs at a particular length in 10−7M NANA can originate from naive CSCCs with SBs that differ in length by 1.0 μm or more (Fig. 3B). From 12% (Fig. 3A) to 24% (Fig. 3B) of the SBs measured before, and after, NANA treatment did not elongate. Although the relative abundance of the different size classes of SBs was arbitrarily adjusted so that the smaller (4 μm) and larger (7–8 μm) SBs were over-represented (Fig. 3B), the results clearly indicate that a significant portion of the SBs do not elongate. An adjusted estimate of these data (Fig. 3B), considering the relative abundance of the different sizeclasses of SBs (e.g., Fig. 4A), suggests that approximately 13 % of SBs selected randomly do not elongate in response to NANA. Thus, estimates of the mean elongation of SBs in response to N-acetylated sugar agonists (Fig. 2) underestimate the increase in length for the elongating SBs.
Ciliary cones are dynamic, vibration-sensitive mechanoreceptors
Four lines of evidence implicate ciliary cones on CSCCs as dynamic, vibration-sensitive mechanoreceptors. (1) Ciliary cones are similar in morphology to known vibrationdetectors such as hair-cell bundles in the acousticolateralis system of vertebrates (Fig. 12B; Bigger, 1982; Marsical et al. 1978; Tilney et al. 1980). (2) Stimulation of chemoreceptors on the supporting cells of CSCCs induces significant and concomitant changes both in the frequency optima for discharging nematocysts (Watson and Hessinger, 1989a) and in the lengths of the SBs of ciliary cones (Figs 2,3,4,7). (3) Both the shift of maximal discharge to lower frequencies and the elongation of SBs adapt to prolonged exposure to 10” ‘M NA.NA, with discharge occurring at essentially the same frequencies as for naive CSCCs (cf. Fig. 6A,C), and SBs shortening to a mean length statistically indistinguishable from the SBs of naive CSCCs (Fig. 4C). Furthermore, upon increasing the NANA concentration for NANA-adapted CSCCs, the optimal frequencies for triggering discharge again shift to lower frequencies (Figs 5D,6D), and the SBs again elongate (Fig. 4D). (4) Treatment in cytochalasin B both disorganizes the SBs of ciliary cones (Fig. 12) and decreases the receptivity of CSCCs to vibrating stimuli (cf. Figs 5,6; 9,10), while having no significant effect on the NANA-sensitized discharge of nematocysts into static test-probes (Fig. 11). Consequently, the effects of cytochalasin treatment on CSCCs appear to be restricted to the reception of vibrating, mechanical stimuli. Thus, it appears that SBs of ciliary cones confer on CSCCs vibration sensitivity for discharging nematocysts.
SB length correlates to frequency-specificity of ciliary cones
As the SBs of CSCCs elongate in response to N-acetylated sugars (Figs 2,3,4,7), the frequency optima for discharging nematocysts shift to lower frequencies (Figs 5,6,8). To establish whether specific size-classes of SBs respond to specific frequencies, we compared dose responses of NANA-induced changes in the abundance of particular size-classes of SBs (Fig. 7) with those of NANA-induced discharge of nematocysts at specific, optimal frequencies (Fig. 8). We stipulated that both (1) the shapes of the dose-response curves and (2) the K0.5 values must be positively correlated to infer that a particular size class of SB is tuned to respond to a specific frequency. On the basis of these comparisons (Table 3), we infer that: (1) SBs measuring 4 μm or smaller are tuned to a frequency of 75 Hz; (2) SBs measuring approximately 5μmin length are timed to a frequency of 55 Hz; and (3) SBs measuring approximately 7 pm in length are timed to a frequency of 30 Hz (Table 3). Furthermore, a single significant peak of nematocyst discharge at 80 Hz was detected for anemones treated with cytochalsin (Figs 9A.10A), and only SBs measuring 3–4 pm were apparent on the tentacle surface. Whether such SBs are shortened remnants of originally longer SBs or a subclass of SBs resistant to cytochalasin treatment, we cannot, as yet, ascertain. The correlations of dose-response parameters suggest that longer mechanoreceptors (SBs) respond to lower frequencies, supporting one model of mechanoreception (Holton and Hudspeth, 1983; Frishkopf and DeRosier, 1984; Howard et al. 1988).
Mechanisms for SB elongation
The mechanism by which the SBs elongate in response to iV-acetylated sugars is not known. The possible mechanisms include those involving alterations in the length of the stereocilia and those involving alterations in the arrangement in the stereocilium bundle. For example, SB elongation could occur by growth of the actin core within each stereocilium, by sliding of the thin filaments within the stereocilia, or by a combination of the two processes. On the other hand, SB elongation could occur by rearranging the stereocilia into a more elongated array without changing the length of the stereocilia. Current research efforts are addressing this issue.
This is the first report of a morphologically dynamic mechanoreceptor in which changes in stereocilium bundle length are coupled to changes in frequency of responsiveness. It furthermore documents that such coupling is under the control of surface chemoreceptors. Thus, ciliary cones of sea anemone CSCCs are morphodynamic, frequency-dynamic and vibration-sensitive mechanoreceptors involved in triggering nematocysts to discharge. The structural and functional dynamism exhibited by these vibration-sensitive mechanoreceptors is regulated by surface chemoreceptors for N-acetylated sugars.
We thank Ms Marcia Kooda, Department of Biology, University of California, Riverside, for technical assistance with scanning electron microscopy and Dr Nancy Mendell, Department of Applied Mathematics and Statistics, SUNY, Stony Brook, for invaluable assistance with statistical analysis. This work was supported by NSF grant DCB 8609859.