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A nematocyst release response in the sea anemone tentacle.

Introduction

Cnidarians are obligate predators. Prey captured by cnidarians are attached to tentacles by discharged nematocysts. The discharged nematocysts, with attached prey, must be released in order for ingestion to proceed (Ewer, 1947). It is not known how the discharged nematocysts are released from the tentacles, and it is not known whether such release is under physiological control. In this paper, we hypothesize a "nematocyst release response." Using methods we have developed to measure the strength of attachment of discharged nematocysts to the tentacles of sea anemones (Thorington and Hessinger, 1996), we experimentally test and measure the nematocyst release response.

We have coined the term "afferent mechanisms" of cnida discharge to refer to those processes acting to or toward the undischarged cnida to regulate or initiate discharge, and to distinguish them from mechanisms acting out of or from the discharged cnida's effector functions, which we have termed "efferent mechanisms" (Thorington and Hessinger, 1996). Cnidae are the eversible secretory products of specialized cells called cnidocytes. The three known classes of cnidae are nematocysts, spirocysts, and ptychocysts (Mariscal, 1974). We have defined and measured an efferent mechanism termed "tentacle adherence." Tentacle adherence indicates how tightly the tentacle holds the capsules of discharged cnidae and is a measure of how tightly targets, such as captured prey, are retained on the tentacles. The force required to remove an average discharged spirocyst or nematocyst from tentacles is the "intrinsic adherence." The intrinsic adherence is calculated from measurements of adhesive force and of the numbers of nematocyst discharged.

We measure adhesive force directly by using a sensitive force-transducer. The adhesive force is the applied force needed to separate a target from the tentacle (Thorington and Hessinger, 1988a). Specifically, adhesive force, as measured from sea anemone tentacles, is the sum of contributions arising from the stickiness of the tentacle mucus to the target ([S.sub.t]) and the product of the number of cnidae (mastigophore nematocysts, [n.sub.m], and spirocysts, [n.sub.s]) discharging onto the target and their intrinsic adherence (mastigophore nematocysts, [i.sub.m], and spirocysts, [i.sub.s]) (Thorington and Hessinger, 1996):

Adhesive force = [S.sub.t] + ([n.sub.m]) ([i.sub.m]) + ([n.sub.s]) ([i.sub.s]) (Equation 1)

Adhesive force and intrinsic adherence may be expressed in units of micronewtons ([Mu]N) or in hybrid units of milligram-force (mgf), since adhesive force is measured without a significant acceleration component and, thereby, does not involve Newton's Second Law (Miller, 1959).

We measured the intrinsic adherence of discharged spirocysts ([i.sub.s]) and of nematocysts ([i.sub.m]) in the sea anemone Aiptasia pallida (Thorington and Hessinger, 1996) and found that the values of [i.sub.s] are consistently very low relative to the values of ira. We concluded that the values of [i.sub.s] are too low to significantly contribute to the measurement of adhesive force or to participate in the retention of struggling prey on feeding tentacles. Thus, equation 1 may be simplified:

Adhesive force = [S.sub.t] + ([n.sub.m]) ([i.sub.m]) (Equation 2)

In anemone tentacles, each cnidocyte is surrounded by two or more supporting cells. The supporting cells possess chemoreceptors (Watson and Hessinger, 1988) and mechanoreceptors (Watson and Hessinger, 1991) that detect prey and trigger nematocyst discharge. The cnidocyte, surrounded as it is by two or more supporting cells, constitutes a cnidocyte/supporting cell complex (CSCC), which we contend is the functional and morphological unit for triggering nematocyst discharge in the sea anemone tentacle.

Two of the three known types of CSCC in sea anemone tentacles are germane to the present study: Types B and C (Thorington and Hessinger, 1990). Some of the CSCCs in anemone tentacles can be made to discharge by mechanical stimulation alone; others require conjoint mechanical and chemical stimulation. We term the former (CSCCs that respond to mechanical stimulation alone) Type C CSCCs; and the latter (which require both chemical and mechanical stimulation) Type B CSCCs. Sensitizing chemoreceptors for N-acetylated sugars (e.g., N-acetylneuraminic acid, NANA) and for certain amino compounds (e.g., glycine, alanine, and proline) have thus far been identified. Stimulation of the chemoreceptors predisposes contact-sensitive mechanoreceptors to respond to contact mechanical stimuli that trigger discharge (Thorington and Hessinger, 1988a; 1990). Type C CSCCs, which are present in lower numbers than Type Bs, do not require chemosensitization but discharge in response to mechanical contact alone.

In addition to proposing the nematocyst release response, we hypothesize that the response is controlled by prey-derived chemicals, as are nematocyst-mediated prey capture (Thorington and Hessinger, 1988b) and the subsequent feeding response (Lindsted, 1971; Lenhoff and Heagy, 1977). Together, our hypotheses predict that certain prey-derived chemicals will lower the intrinsic adherence of discharged nematocysts.

Using methoxyverapamil (D-600) to selectively inhibit discharge from Type Bs, we show that (i) NANA inhibits the intrinsic adherence of nematocysts discharged from Type C CSCCs and, therefore, controls the release response of Type Cs; and (ii) glycine inhibits the intrinsic adherence of nematocysts discharged from Type B CSCCs and, therefore, controls the release response of Type Bs. We conclude that a nematocyst release response exists in sea anemones and that it is controlled by prey-derived chemicals that also control prey capture.

Materials and Methods

Maintenance of sea anemones

Monoclonal sea anemones (A. pallida, Carolina strain) were maintained individually in glass finger bowls containing natural seawater at 24 [degrees] [+ or -] 1 [degrees] C as previously described (Hessinger and Hessinger, 1981; Thorington and Hessinger, 1988a). Briefly, anemones were fed daily with freshly hatched brine shrimp nauplii (Anemia salina) and washed 4-6 h after feeding (Hessinger and Hessinger, 1981). Anemones were maintained on a 12/12-h photoperiod using white fluorescent lights at an intensity of 5.5 klux (66 [micro][Es.sup.-1][m.sup.-2]). Animals were starved for 72 h prior to experiments.

Experimental animals and test solutions

Filtered, natural seawater was obtained from Kerckoff Marine Laboratory of California Institute of Technology at Corona del Mar, California. Animals of same size were starved 72 h prior to experimentation and kept under constant fluorescent light at 4.5 klux (54 [Mu][Es.sup.-1][m.sup.-2]) during the last 48 h of starvation. Exposure to continuous light enhanced the uniformity of anemone behavior and cnidocyte responsiveness. Just prior to use, the animals were gently rinsed to remove soluble waste, and the medium was replaced with test solutions. Unless otherwise stated, animals were permitted to adapt to the change of medium for 10 min before cnidocyte responsiveness was measured. N-acetylneuraminic acid (NANA), glycine, and methoxyverapamil (D-600) were obtained from Sigma Chemical Co., St. Louis, Missouri. Solutions of D-600 were made up fresh on the day of the experiment and protected from light. All test solutions were prepared in artificial seawater (ASW) adjusted to pH 7.62 with 1 N HCI or NaOH. The ASW consisted of 423 mM NaCl, 10 mM KCl, 24 mM Mg[Cl.sub.2], 25 mM MgS[O.sub.4], 10 mM Ca[Cl.sub.2], and 1.2 mM NaHC[O.sub.3]. Calcium-free artificial seawater (Ca-free ASW) was prepared with the same components as ASW except that calcium chloride was omitted, the NaCl concentration was increased to 438 mM, and EGTA (ethyleneglycoltetraacetic acid) was added to a final concentration of 1 mM; and then the final pH was adjusted to 7.62.

Assays of cnidocyte responsiveness

Physical contact of a tentacle with a gelatin-coated probe triggers discharge of local nematocysts and spirocysts, and adherence of the tentacle to the probe (Thorington and Hessinger, 1988b, 1990). Four parameters were measured to analyze nematocyst-mediated adhesive force: total adhesive force; number of discharged nematocysts; number of discharged spirocysts; and adhesive force in tentacles in which nematocyst and spirocyst discharge had been inhibited by pretreatment with formaldehyde to measure tentacle stickiness. The methods have been described in detail previously (Thorington and Hessinger, 1990). The number of cnidae on the probes is a direct measure of the number of cnidae discharged.

Measurement of adhesive force. Cnida-mediated adhesive force was measured as previously described (Thorington and Hessinger, 1988a). This technique involves using small gelatin-coated nylon beads of defined diameter attached to a strain gauge by means of a fine stainless steel shaft. The gel-coated bead is made to contact the distal third of a primary tentacle on an anemone in a finger bowl containing the test solution. The discharge of cnidae initiated by contact of the probe with the tentacle causes the tubules of everting cnidae to either adhere to or penetrate the gelatin surface. Withdrawing the probe from the tentacle causes the discharged cnidae to exert an opposing force on the probe; this force is measured with a gravimetrically calibrated force-transducer connected to a potentiometric recorder. The force necessary to separate the probe from the tentacle is called the adhesive force and is expressed in hybrid units of milligram-force (mgf). It is an aggregate measure of the "inherent" stickiness of the tentacle plus the nematocyst-mediated adhesive force.

Counting discharged nematocysts. After adhesive force measurements, the same probes are processed for counting nematocysts as detailed previously (Geibel et al., 1988). Briefly, the gelatin coating of the probes is enzymatically digested to release the nematocysts of the discharged mastigophores. The highly refractive mastigophores, which are resistant to proteolysis, are then counted with an inverted microscope from the fiat bottoms of microtiter wells.

Counting discharged spirocysts. To determine the number of discharged spirocysts, we used an indirect, solid-state enzyme-linked lectin sorbant assay (ELLSA), the details of which have been published (Thorington and Hessinger, 1990). In principle, the assay is based upon the high affinity of conjugated N-acetylated sugars to the everted tubules of discharged spirocysts on the surface of the test probes. The assay involves use of a microtiter-plate spectrophotometer for colorimetric determination of bound peroxidase activity after the sequential treatment of test probes with solutions of asialomucin and Vicia villosa lectin/peroxidase conjugate.

Collection and analysis of data

Individual animals were tested at each concentration of sensitizer. Twelve probes (one per tentacle) were used on each animal to determine adhesive force and to count discharged nematocysts or spirocysts. Daily experimental means were calculated from these experiments. Replicate experiments were carried out on three different days. Each data point represents the mean of the three daily experimental means, and the range bar represents the standard error of the mean.

Results

Selective discharge of cnidae from Type C cnidocyte/supporting cell complex

To test our hypotheses that a nematocyst release response exists and is under the control of prey-derived chemicals, we ask the research question: Do known chemosensitizers of nematocyst discharge lower, in a dose-dependent manner, the values of [i.sub.m] for nematocysts discharged from either Type B or Type C CSCCs? To answer this question we proposed to determine the intrinsic adherence (ira values) for nematocysts discharged from Type B and from Type C CSCCs as a function of chemosensitizer concentration. We selectively blocked chemosensitized discharge of nematocysts from Type B CSCCs by using 2-methoxyverapamil (D-600) (Thorington and Hessinger, 1992; Watson and Hessinger, 1994b), a diphenylalkylamine inhibitor of vertebrate L-type calcium channels (Spedding and Paoletti, 1992). With discharge from Type B CSCCs blocked, we directly determined the effect of chemosensitizers on the intrinsic adherence of nematocysts discharged from Type Cs. By subtracting the responses of the Type Cs from the combined responses of Type B and Type C CSCCs (in the presence of chemosensitizer, but the absence of D-600), we calculated the intrinsic adherence of nematocysts from the Type Bs.

D-600 inhibits discharge from Type B CSCCs. D-600 potently and dose-dependently inhibits nematocyst discharge from NANA-sensitized Type Bs ([ILLUSTRATION FOR FIGURE 1 OMITTED]; open circles). D-600 also inhibits glycine-sensitized nematocyst discharge ([ILLUSTRATION FOR FIGURE 1 OMITTED]; open squares), but less potently and over a wider range of D-600 concentrations. In the absence of chemosensitizers, mechanical contact elicits discharge only from Type C CSCCs (Thorington and Hessinger, 1990). D-600 has no detectable effect on nematocyst discharge from Type Cs at any tested dose ([ILLUSTRATION FOR FIGURE 1 OMITTED]; closed circles). The half-inhibitory doses ([IC.sub.50]) for D600 on NANA- and glycine-sensitized discharge is below [10.sup.-16] M for NANA and [10.sup.-15] M for glycine, and the minimal doses that maximally inhibit ([IC.sub.100]) sensitized discharge are about [10.sup.-10] M for NANA and [10.sup.-6] M for glycine. Thus, D-600 blocks nematocyst discharge from Type B CSCCs, but not from Type Cs.

D-600 lowers tentacle stickiness ([S.sub.t]). We tested the effects of D~600 on tentacle stickiness (Table I) by incubating anemones at room temperature in ASW and in Ca-free ASW with and without D-600. After 20 min we added formalin to 10% for 5 min to measure [S.sub.t] with adhesive force probes in the absence of any cnida discharge. The value of [S.sub.t] is not altered by 10% formalin (Thorington and Hessinger, 1996). As expected, neither nematocysts nor spirocysts are discharged in 10% formalin (Table I). The mean value of St in ASW is 36.6 [+ or -] 0.9 mgf (358.7 [+ or -] 8.8 [Mu]N; Table I), which is in close agreement with the value of 34.6 [+ or -] 0.7 mgf (339.1 [+ or -] 6.9 [Mu]N) that we previously reported for similar experimental conditions (Thorington and Hessinger, 1996). The values of St are decreased 44% from ASW controls by [10.sup.-10] M D-600 in ASW (P [less than] 0.001) and 49% from ASW controls by [10.sup.-8] M D-600 in ASW (P [less than] 0.005). In Ca-free ASW, the mean value of St is 31.3 [+ or -] 0.9 mgf (306.7 [+ or -] 8.8 [Mu]N, a value 15% lower (P [less than] 0.02) than in ASW containing 10 mM [Ca.sup.2+]; the combination of Ca-free ASW and [10.sup.-10] M D-600 causes a decrease of 55% from ASW (P [less than] 0.001; Table I). Thus, D-600 significantly and dose-dependently lowers tentacle stickiness ([S.sub.t]).

Effects of N-acetylneuraminic acid on intrinsic adherence

The combined dose-responses of Type B and Type C CSCCs to NANA for nematocyst discharge, spirocyst discharge, and adhesive force are characteristically biphasic ([ILLUSTRATION FOR FIGURES 2a-c OMITTED], open circles; Thorington and Hessinger, 1990). In the presence of [10.sup.-10] M D-600, the dose-responses of Type Cs to NANA for nematocyst and spirocyst discharge are level and at control levels, showing no significant changes ([ILLUSTRATION FOR FIGURES 2a AND b OMITTED], closed circles). Thus, discharge from Type B CSCCs to NANA is totally inhibited by [10.sup.-10] M D-600, whereas discharge from Type Cs is unaffected.

NANA lowers [Mathematical Expression Omitted]. Nematocyst-mediated adhesive force is calculated by subtracting tentacle stickiness ([S.sub.t]) from adhesive force. To obtain nematocyst-mediated adhesive force in dose-responses to NANA, we subtracted from adhesive force measurements the appropriate value of [S.sub.t] from Table I depending upon whether D-600 was or was not present during the experimental measurements. Nematocyst-mediated adhesive force from Type Cs steadily declines as the NANA concentration increases ([ILLUSTRATION FOR FIGURE 2c OMITTED], closed circles). Since the numbers of nematocysts and spirocysts discharging from Type Cs do not significantly decrease ([ILLUSTRATION FOR FIGURES 2a AND b OMITTED], closed circles), the decline in adhesive force is probably due to a decrease in the value of [i.sub.m] from the Type C CSCCs (i.e., [[i.sub.m].sup.c]).

We calculated the [i.sub.m] values of nematocysts discharging from Type Cs (i.e., [[i.sub.m].sup.c]) across a wide range of NANA concentrations ([10.sup.-16] to [10.sup.-3] M; [ILLUSTRATION FOR FIGURE 2d OMITTED], closed circles) by using the values for nematocyst discharge and nematocyst-mediated adhesive force of Type C CSCCs in Equation 2. The values of [[i.sub.m].sup.c] decrease with increasing levels of NANA, from about 0.20 mgf (1.96 [Mu]N) in ASW to [TABULAR DATA FOR TABLE I OMITTED] near zero at [10.sup.-4] M NANA. The concentration of NANA that half-inhibits [[i.sub.m].sup.c] is approximately [10.sup.-12] M NANA. Thus, NANA dose-dependently lowers the intrinsic adherence of nematocysts discharged from Type Cs.

NANA biphasically modulates [[i.sub.m].sup.b]. The dose-responses for discharge and adhesive force from Type Bs ([ILLUSTRATION FOR FIGURE 2ac OMITTED], closed squares) are calculated by subtracting the discharge (or adhesive force) of Type Cs in D-600 (closed circles) from the discharge (or adhesive force) of both Type Bs and Cs in the absence of D-600 (open circles). The calculated Type B dose-responses of both mastigophore and spirocyst discharge are narrow and biphasic. Maximum discharge ([E.sub.max]) of nematocysts and spirocysts from Type Bs is about two times and one time, respectively, that of Type C ASW controls, suggesting that Type Bs and Type Cs coexist in the tentacle in ratios of 2:1 and 1:1 for nematocyst-bearing and spirocyst-bearing CSCCs, respectively. The concentration at which maximal discharge occurs ([EC.sub.100]) is about [10.sup.-5] M NANA, and the concentration at which half-maximum discharge occurs ([K.sub.0.5]) is about [10.sup.-6] M NANA. The NANA dose-response of nematocyst-mediated adhesive force from Type Bs is broadly biphasic, with an [EC.sub.100] of [10.sup.-5] M NANA and a [K.sub.0.5] of about [10.sup.-10] M NANA ([ILLUSTRATION FOR FIGURE 2c OMITTED], open circles).

Using the values for nematocyst discharge and nematocyst-mediated adhesive force of Type B CSCCs in Equation 2, we calculate the [i.sub.m] values of nematocysts discharging from Type Bs (i.e., [[i.sub.m].sup.b]) across a wide range of NANA concentrations ([ILLUSTRATION FOR FIGURE 2d OMITTED], closed squares). The [[i.sub.m].sup.b] dose-response to NANA is biphasic, with maximum [[i.sub.m].sup.b] values of about 0.8 mgf (7.8 [Mu]N) occurring at [10.sup.-10] M NANA and the [K.sub.0.5] occurring at about [10.sup.-12] M NANA. The peak of the [[i.sub.m].sup.b] dose-response is about five orders of magnitude lower than the peak of the dose-response of Type B nematocyst discharge [ILLUSTRATION FOR FIGURE 2a OMITTED]. We conclude that NANA dose-dependently modulates a bipbasic change in the intrinsic adherence of nematocysts discharging from Type B CSCCs, but does not reduce the value of [[i.sub.m].sup.b] to zero.

Effects of glycine on intrinsic adherence

Glycine suppresses discharge from Type Cs. The minimum dose of D-600 that totally inhibits glycine-sensitized nematocyst discharge from Type Bs is [10.sup.-6] M [ILLUSTRATION FOR FIGURE 1 OMITTED]. To minimize possible side effects from such a relatively high dose, we elected to use [10.sup.-8] M D-600 rather than [10.sup.-6] M. The dose-responses to glycine for nematocyst discharge, spirocyst discharge, and adhesive force are characteristically biphasic ([ILLUSTRATION FOR FIGURES 3a-c OMITTED]; Thorington and Hessinger, 1990). In [10.sup.-8] M D-600 the dose-responses to glycine for discharge of both nematocysts and spirocysts and for adhesive force do not exceed those of controls ([ILLUSTRATION FOR FIGURES 3a-c OMITTED], closed circles). Thus, [10.sup.-8] M D-600 appears to inhibit cnida discharge from Type B CSCCs in response to glycine.

On the other hand, in the presence of [10.sup.-8] M D-600, we observe about a 50% decrease in both nematocyst and spirocyst discharge from Type Cs at the lowest tested doses of glycine (i.e., [10.sup.-16] M glycine). Nematocyst discharge gradually recovers to control levels of discharge at about [10.sup.-7] M glycine and then declines again at higher concentrations. Spirocyst discharge recovers at lower concentrations of glycine and reaches control levels at [10.sup.-6] M glycine before it, like nematocyst discharge, declines again at higher concentrations. Thus, low concentrations of glycine suppress both nematocyst and spirocyst discharge from Type C CSCCs.

Glycine modulates [[i.sub.m].sup.c]. Nematocyst-mediated adhesive force from Type Cs remains constant (between 12 and 15 mgf or 118 and 147 [Mu]N) at all tested glycine concentrations except [10.sup.-7] M glycine, at which concentration the adhesive force increases to 20 mgf (196 [Mu]N; [ILLUSTRATION FOR FIGURE 3c OMITTED]; closed circles). Using Equation 2, we calculated the [i.sub.m] values of nematocysts discharging from Type Cs (i.e., [[i.sub.m].sup.c]) across the range of tested glycine concentrations ([10.sup.-16] to [10.sup.-4] M; [ILLUSTRATION FOR FIGURE 3d OMITTED], closed circles). The values of [[i.sub.m].sup.c] are relatively high (about 0.6 mgf/nematocyst or 5.9 [Mu]N/nematocyst) in seawater controls and in [10.sup.-16] to [10.sup.-12] M glycine. But between [10.sup.-10] and [10.sup.-7] M glycine, the values of [[i.sub.m].sup.c] decrease by about one-half (to about 0.3 mgf/nematocyst or 2.9 [Mu]N/nematocyst) and then increase (to about 0.4 mgf/nematocyst or 3.9 [Mu]N/nematocyst) at [10.sup.-6] M and higher concentrations. Overall, there appears to be a downward trend in [[i.sub.m].sup.c] with increasing glycine concentrations.

Glycine suppresses [[i.sub.m].sup.b]. In contrast to NANA, in which NANA-sensitized discharge of nematocysts from Type Bs spans a narrow range of NANA concentrations ([ILLUSTRATION FOR FIGURE 2a OMITTED], closed squares), glycine-sensitized discharge of nematocysts spans a wide range of glycine concentrations ([ILLUSTRATION FOR FIGURE 3a OMITTED], closed squares). At all tested glycine concentrations (except [10.sup.-16] M), the numbers of nematocysts discharged from Type Bs exceed those discharged from Type Cs. Yet the values of [[i.sub.m].sup.b] in glycine never exceed 0.1 mgf/nematocyst (0.98/[Mu]N/nematocyst; [ILLUSTRATION FOR FIGURE 3d OMITTED], closed squares), unlike the values in NANA which never drop below 0.2 mgf/nematocyst (1.96 [Mu]N/nematocyst; [ILLUSTRATION FOR FIGURE 2d OMITTED], closed squares).

Discussion

Cnidarians capture swimming prey by discharging nematocysts from their tentacles. These same nematocysts also secure the struggling prey to the tentacle during coordinated tentacle movements that transport the prey to the oral disk and mouth. For ingestion to occur, however, the nematocysts securing captured prey to the tentacle must then be released from the tentacle (Ewer, 1947).

We proposed a so-called nematocyst release response controlled by soluble, prey-derived chemicals also known to both chemosensitize nematocyst discharge and initiate the feeding response. We stipulated that the strength of attachment of discharged nematocysts to sea anemone tentacles (i.e., intrinsic adherence) must be reduced to or near zero for the mechanism to qualify as a nematocyst release response.

Effects of N-acetylneuraminic acid and glycine on discharge and intrinsic adherence

NANA does not affect discharge from Type Cs. Because low concentrations of D-600 block both NANA-sensitized and glycine-sensitized discharge from Type B CSCCs without affecting discharge from Type Cs [ILLUSTRATION FOR FIGURES 1 AND 2A, B OMITTED], we were able to measure the response from Type Cs without interfering contributions from discharging Type Bs. Throughout the range of [10.sup.-16] to [10.sup.-3] M NANA, nematocyst and spirocyst discharge from Type Cs was constant [ILLUSTRATION FOR FIGURES 2a, b OMITTED]. We concluded that neither nematocyst nor spirocyst discharge from Type Cs is affected by NANA chemoreceptor stimulation.

NANA controls the release response of Type Cs. Despite constant nematocyst discharge from Type Cs at all tested NANA concentrations, nematocyst-mediated adhesive force from Type Cs declined with increasing NANA concentrations [ILLUSTRATION FOR FIGURE 2c OMITTED]. The decline in adhesive force was caused by a dose-dependent decrease (from 0.2 to 0 mgf/nematocyst, or 1.96 to 0 [Mu]N/nematocyst; [ILLUSTRATION FOR FIGURE 2d OMITTED]) in the intrinsic adherence of nematocysts discharged from Type Cs ([[i.sub.m].sup.c]). The dose-dependent decrease in [[i.sub.m].sup.c] fits our criterion of a nematocyst release response. We concluded that a release response of nematocysts discharged from Type Cs is under the control of NANA chemoreceptors.

NANA controls [[i.sub.m].sup.b]. We calculated the NANA dose-responses of Type B CSCCs by subtracting NANA dose-responses in the presence of [10.sup.-10] M D-600 from NANA dose-responses in the absence of D-600 [ILLUSTRATION FOR FIGURE 2 OMITTED]. The calculated NANA dose-responses of both nematocyst and spirocyst discharge from Type Bs were narrow and bipbasic, with maximal discharge occurring at [10.sup.-5] M NANA ([EC.sub.100]) and half-maximal discharge ([K.sub.0.5]) occurring at [10.sup.-6] M NANA [ILLUSTRATION FOR FIGURES 2a, b OMITTED]. The calculated NANA dose-response of nematocyst-mediated adhesive force from Type Bs was also biphasic, but over a much broader range of NANA concentrations, with the [EC.sub.100] still occurring at [10.sup.-5] M NANA, but with the [K.sub.0.5] occurring much lower (at [10.sup.-10] M NANA; [ILLUSTRATION FOR FIGURE 2c OMITTED]).

In NANA, the intrinsic adherence of nematocysts discharged from Type Bs ([[i.sub.m].sup.b]) varied biphasically with NANA concentration [ILLUSTRATION FOR FIGURE 2d OMITTED]. The maximal [[i.sub.m].sup.b] value of 0.8 mgf/nematocyst (7.8 [Mu]N/nematocyst) occurred at [10.sup.-10] M NANA, but decreased to about 0.2 mgf (1.96 [Mu]N) at [10.sup.-5] M NANA and at higher concentrations. The values of [[i.sub.m].sup.c] in NANA varied from 0.2 to 0 mgf/nematocyst (1.96 to 0 [Mu]N/nematocyst). Thus, in seawater containing NANA, [[i.sub.m].sup.b] will exceed [[i.sub.m].sup.c]. It therefore appears that in the presence of NANA, nematocysts discharged from Type Bs will predominate in both prey capture and prey retention over nematocysts discharged from Type Cs. Although the NANA dose-response of [[i.sub.m].sup.b] never goes to zero, we conclude that NANA controls [[i.sub.m].sup.b] over a wide range of values, in a dose-dependent manner, but without affecting the release response of nematocysts discharged from Type Bs.

Glycine inhibits discharge from Type Cs. In addition to N-acetylated sugars, amino sensitizers, such as glycine, also predispose nematocysts and spirocysts to discharge in response to mechanical stimuli (Thorington and Hessinger, 1988a, 1990). In the present study, we showed that glycine sensitizes Type B CSCCs to discharge nematocysts and spirocysts [ILLUSTRATION FOR FIGURES 3a, b OMITTED]. However, unlike NANA, which had no effect on discharge from Type Cs, glycine suppressed nematocyst and spirocyst discharge from Type Cs [ILLUSTRATION FOR FIGURES 3a, b OMITTED]. The inhibitory effect of glycine was partial and occurred at both low and high concentrations, but reversed at intermediate concentrations. The inhibitory effect of glycine on discharge from Type Cs is probably different from the inhibitory effects of certain homogenate fractions of Anemia on prey capture in Hydra (Grosvenor and Kass-Simon, 1987) since the inhibitory action of glycine in Aiptasia is restricted to Type Cs and the net effect of glycine is to increase nematocyst discharge due to its sensitizing effect on the more numerous Type Bs [ILLUSTRATION FOR FIGURE 3a OMITTED]. To the best of our knowledge, this is the first report of an identified chemoreceptor inhibiting nematocyst discharge.

The value of [[i.sub.m].sup.c] varied from 0.6 mgf/nematocyst (5.9 [Mu]N/nematocyst; in seawater and in lower glycine concentrations) to about 0.3 and 0.4 mgf/nematocyst (2.9 and 3.9 [Mu]N/nematocyst; at higher glycine concentrations). Although NANA lowers the [[i.sub.m].sup.c] to zero at higher concentrations, thereby constituting a nematocyst release response, glycine also lowers [[i.sub.m].sup.c], but without bringing the value to zero. Glycine inhibits discharge from Type Cs, but it does not affect the Type C nematocyst release response.

Glycine controls the release response of Type Bs. Although glycine-sensitized nematocyst discharge from Type Bs is robust at all concentrations greater than [10.sup.-14] M, the value of [[i.sub.m].sup.b] is consistently low, never exceeding values of 0.1 mgf/nematocyst and reaching 0 mgf/nematocyst (0.98 and 0 [Mu]N/nematocyst) at [10.sup.-4] M glycine. We conclude that the action of glycine on the intrinsic adherence of nematocysts discharged from Type B CSCCs is consistent with glycine control of the release response of nematocysts discharged from Type Bs.

D-600 affects Type Bs differently than Type Cs. The inhibition of nematocyst discharge from Type B CSCCs by verapamil and other organic and inorganic inhibitors of voltage-gated calcium channels was first reported in the anemone Haliplanella luciae (Watson and Hessinger, 1994a). We now show that D-600, a water-soluble derivative of verapamil, inhibits both nematocyst and spirocyst discharge from Type Bs in Aiptasia pallida [ILLUSTRATION FOR FIGURE 2A, B OMITTED]. The effects on nematocyst discharge of calcium depletion and of standard organic and inorganic calcium channel blockers (Watson and Hessinger, 1994a) are consistent with the involvement of calcium channels, pharmacologically resembling L-type calcium channels, in the chemosensory signaling pathway of Type B CSCCs. Our finding that NANA-sensitized discharge of nematocysts is much more sensitive to D-600 than is glycine-sensitized discharge [ILLUSTRATION FOR FIGURE 1 OMITTED] suggests that the NANA and glycine chemosensory pathways utilize calcium in different ways.

Both D-600 and calcium-free seawater reduce tentacle stickiness, and these effects are additive (Table I), suggesting common modes of action. Tentacle stickiness is mediated by the interaction of the mucus layer covering the tentacle surface with the probe. Tentacle mucus originates, in part, by a constitutive secretory process of apical secretory cells. Under conditions of injury or stress, the amount of surface mucus increases. Thus, tentacle mucus may also be contributed by regulated secretion. The nematocyst is a secretory product of the cnidocyte (Slautterback, 1961; Skaer, 1973). Exocytotic processes (i.e., mucus secretion and nematocyst discharge [Holstein and Tardent, 1984]), in general, require extracellular calcium and calcium influx mediated by calcium channels (Bennett et al., 1979). We hypothesize that D-600 and calcium-free seawater reduce tentacle stickiness and inhibit discharge from Type B CSCCs by affecting involved calcium channels.

Roles of N-acetylneuraminic acid and glycine in prey capture and prey ingestion

Prey capture. In our view the "NANA" receptor is the primary chemoreceptor for capturing prey since N-acetylated sugars are ubiquitous constituents of the surfaces of most aquatic prey. On the other hand, the activators of the "amino" chemoreceptors (glycine, alanine, and proline) occur in high concentrations in the hemolymph of crustacean prey (Gilles, 1979) and of Artemia nauplii (Clegg and Conte, 1980), the typical prey of laboratory-reared cnidarians. Presumably the amino sensitizers leak into the ambient medium from wounds inflicted by NANA-sensitized nematocysts, as originally suggested by Loomis of the glutathione-effected feeding response of Hydra (Loomis, 1955) and later confirmed by Lenhoff (1961). Proline, for instance, potently and negatively modulates NANA-induced tuning of vibration-sensitive mechanoreceptors associated with Type A CSCCs in a related anemone, Haliplanella luciae (Watson and Hessinger, 1994b). Alanine inhibits NANA-sensitized discharge from Type Bs in Aiptasia (unpubl. findings). And glycine inhibits discharge of nematocysts and spirocysts from Type C CSCCs [ILLUSTRATION FOR FIGURE 3A, B OMITTED]. Thus, we view the "amino" receptors to be secondary chemoreceptors in prey capture, involved in negatively modulating the primary prey capture response to N-acetylated sugars. As secondary chemoreceptors, the amino receptors respond to chemicals leaking from wounded prey and assure that only the minimum adequate number of nematocysts are discharged. By conserving nematocysts and avoiding "overkill," the anemone devotes fewer prey nutrients to replacement of nematocysts and more to growth and reproduction.

Prey ingestion. Following prey capture and transport to the mouth, glycine from wounded prey acts in concert with NANA to stimulate the release responses of discharged nematocysts so that prey can be ingested. Our present work shows that NANA controls the release response of nematocysts discharged from Type Cs [ILLUSTRATION FOR FIGURE 2D OMITTED], whereas glycine controls the release response of nematocysts from the more numerous Type Bs [ILLUSTRATION FOR FIGURE 3D OMITTED].

It has been observed, although not, to our knowledge, published, that many cnidarians do not ingest dead prey, even though the dead prey elicit nematocyst discharge and adhere to tentacles (e.g., pers. obs. by C. Hand, UC, Davis; K. Heidelberg, U. Maryland; H. M. Lenhoff, UC, Irvine). Discrimination between living and dead prey appears to occur after prey capture and may involve the detection of amino substances leaking from wounded prey. On the one hand, the very short timescale for a successful nematocyst response dictates that chemical discrimination in prey capture be cursory and based upon a single chemical cue, the external N-acetylated sugars. These sugars are common to living and dead prey, so discrimination is sacrificed for response speed and the drifting remains of dead prey containing no nutrients are occasionally captured. On the other hand, the subsequent release response and prey ingestion take place on a much longer timescale than prey capture. The release response is based upon dual chemosensory cues, N-acetylated sugars and glycine. Dead and nutrient-depleted prey, if captured, will not provide the amino chemosensory cue needed to stimulate a complete nematocyst release response, and such prey will not be ingested. The dual chemoreceptor requirement for the release response ensures that captured dead prey are not ingested and digestive resources are not committed to nutrient-poor prey.

The [i.sub.m]-discharge plot explained

We showed previously (Thorington and Hessinger, 1996) that a plot of the weighted average [i.sub.m] values versus the total number of discharged nematocysts (the so-called [i.sub.m]-discharge plot) was inversely hyperbolic in shape for NANA and several amino chemosensitizers. Except for the steepness of the [i.sub.m]-discharge plot, our dilution/recruitment model, in general, simulated the plot. The dilution/recruitment model assumed that (i) the [i.sub.m] values of mastigophore nematocysts from both Type B and Type C CSCCs are constant, (ii) the value of [[i.sub.m].sup.c] is larger than the value of [[i.sub.m].sup.b], and (iii) the number of nematocysts discharged from Type C CSCCs is constant, (iv) while the number discharged from Type B CSCCs varies biphasically with increasing concentrations of chemosensitizer. In this model the discharge of a constant number of high-[i.sub.m] nematocysts from Type C CSCCs is numerically diluted by the discharge of progressively greater numbers of lower-[i.sub.m] nematocysts recruited by chemosensitization of Type B CSCCs.

Our present findings indicate that the recruitment/dilution model is incomplete in several ways. Firstly, the [i.sub.m] values from Type Bs and Cs are not constant. We have shown that NANA biphasically varies [[i.sub.m].sup.b] and glycine monophasically varies [[i.sub.m].sup.c]. Secondly, the values of [[i.sub.m].sup.c] are not always larger than the values of [[i.sub.m].sup.b]. We have found that [[i.sub.m].sup.b] is greater than [[i.sub.m].sup.c] in the presence of NANA. Thirdly, the number of nematocysts discharged from Type C CSCCs is not always constant. In the presence of glycine the number of nematocysts discharging from Type Cs decreases. On the positive side, our assumption that the numbers of nematocysts discharged from Type B CSCCs varies biphasically with increasing concentrations of chemosensitizer was confirmed for both NANA and glycine.

Conclusions

1. Nematocysts perform at least three feeding-related functions. (i) Discharging nematocysts subdue swimming prey by penetrating and envenomating prey. (ii) Discharged nematocysts secure straggling prey to the tentacles by a combination of barbed nematocyst tubules fastened into the integument of prey and the initially high intrinsic adherence of nematocyst capsules' attachment to the tentacle. (iii) Discharged nematocysts subsequently facilitate prey ingestion by being released from the tentacle as a result of a large decrease in intrinsic adherence.

2. Each of the feeding-related functions of nematocysts is under the control of at least two chemoreceptor systems. One system is for N-acetylated sugars, such as N-acetylneuraminic acid (NANA), which occur on the surface of prey; the other is for "amino" substances, such as glycine, which occur in the blood of prey and are leaked into the medium from nematocyst-inflicted wounds.

3. The "NANA" receptor is the primary chemoreceptor controlling prey capture, while the "amino" receptors act to negatively modulate nematocyst discharge. Glycine, for instance, inhibits the discharge of nematocysts (and spirocysts) from Type C CSCCs over a wide range of concentrations.

4. To ensure that captured prey are tightly secured to the tentacle, the "NANA" receptor raises the intrinsic adherence of nematocysts discharged from Type B CSCCs at low concentrations of NANA (about [10.sup.-10] M).

5. To ensure that captured prey are subsequently ingested, the nematocyst release response is activated for nematocysts discharged from Type C CSCCs and from Type B CSCCs by higher concentrations ([10.sup.-4] M) of NANA and glycine, respectively.

6. Cnida-independent tentacle stickiness, presumably due to surface mucus, is dose-dependently decreased by D-600 and by removal of extracellular calcium. The inhibitory actions of D-600 and low calcium on tentacle stickiness are additive.

Acknowledgments

Supported in part by NSF grant MCB-8919269.

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Title Annotation:Efferent Mechanisms of Discharging Cnidae, part 2
Author:Thorington, Glyne U.; Hessinger, David A.
Publication:The Biological Bulletin
Date:Oct 1, 1998
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