Factors governing nematocyst discharge from cnidophage celis of the nudibranch Aolidea papillosa Catherine L. Gallagher Stanford Department of Biological Sciences Hopkins Marine Station Pacific Grove, California 93950 0 My thanks to Rob Swezey for his advice and humorous comments Factors Governing Nematocyst Discharge from Cnidophage Cells of the Nudibranch Aeolidea papillosa Catherine L. Gallagher Department of Biological Sciences, Stanford University Hopkins Marine Station Pacific Grove, CA 93950 Abstract Nemacysts isolated from Aeolidea papillosa were subjected to different ionic media and their frequency of discharge assessed. Discharge of nematocysts isolated by homogenization could not be induced, suggesting that proper function of the cnidae is at least partially dependent on surrounding tissues. When nematocysts are extruded from intact cerata into ionic media, conditions that inhibit normal contraction of the cerata also reduce nematocyst discharge. Some aspect of passage through the cnidopore or contact with sea water could initiate an intracellular signal in the cnidophage that causes its nematocysts to discharge. A battery of second-messenger inducing systems including forskolin (stimulates adenylate cyclase), TPA (stimulates protein kinase C, calcium ionophore (causes release of intracellular Ca stores), and pH affectors failed, however, to cause nematocyst discharge from isolated cnidophage cells. Introduction Cnidarians, including sea anemones and hydra, are endowed with specialized stinging organelles called cnidae. Three types of cnidae¬ spyrocysts, nematocysts, and ptychocysts- reside in specialized cells, the cnidocytes, lining the tentacles. Nematocysts contain a coiled tubule that everts under excitatory conditions to produce a thin thread or barbed tubule through which poison may be dispatched. Eversion of the tubule has been classified as rapid excocytosis and occurs in less than three milliseconds. (Holstein and Tardent, 1984) Spyrocysts, on the other hand, discharge a fine web that clings to prey items, holding them close to the tentacles where they can be pierced by nematocysts. Neither the direct cause of nematocyst excitation nor the mechansim of eversion is well understood. Three models have been proposed to explain nematocyst discharge: (1) In response to some stimulus, a sudden osmotic change in the contents of the capsule causes an influx of vater and the sudden pressure forces ejection of the tubule. Measurements of the capsular contents show that thread eversion is accompanied by a dramatic decrease in calcium concentration within the capsule. (Lubbock and Amos, 1981) Considering the relatively high amounts of calcium (600 mM) present in the undischarged capsule, researchers speculate that this calcium binds soluble proteins into aggregates. Upon some stimulus calcium is removed from these complexes, releasing the proteins into solution thereby increasing the osmolarity of the intracapsular fluid (Lubbock and Amos, 1981). The subsequent influx of water causes discharge. (2) A second hypothesis postulates that the three apical flaps at the active end of the capsule prevent or permit eversion. Packaged like a coiled spring, the thread is constantly straining to straighten itself and any disruption of the apical flaps that hold it in place allows it to shoot out. Watson and Mariscal (1985) propose that binding of an attached protein to calcium holds the flap in place. Departure of calcium, therefore, causes a loostening of the flaps and the tubule everts. (3) It has been suggested that contractile action of fibers in the capsule or in surrounding structures causes the tubule to squirt out. (D. Hessinger. personal communication) As unresolved as the mechanism of discharge is the identity of stimuli that initiate the process. Blanquet (1970) found that nematocysts isolated from sea anemones appeared to be stabilized against discharge by divalent cations. particularly Mg- and Ca*; high concentrations of K', on the other hand, caused them to discharge more readily than in sea water. Having acquainted myself with the behavior of nematocysts from the anemone, I decided to compare this system with that of the aolid nudibranch. These nudibranchs store nematocysts from their cnidarian prey in specialized gut projections called cerata and release them for defense against predators. The cerata extend dorsally, each terminating in a specialized structure called the cnidosac. (Figure 1) A sphincter muscle forms a narrow channel separating the cnidosac from the rest of the gut. Nematocysts pass into the cnidosac where they are endocytosed by specialized cells called cnidophages. When alarmed, the nudibranch releases cnidophages through a pore in the ceras tip called the cnidopore and nematocysts discharge. Whether the pore preexists or is produced by rupture of the epithelium remains controversial (Greenwood and Mariscal, 1984). What conditions does the nudibranch have to provide for its acquired cnidae so that they function properly? Do the nematocysts simply discharge upon contact with sea water when the cnidophage membrane ruptures, as suggested by Greenwood and Mariscal, (1984), or is the system more complicated? Could a second messonger within the cnidophage coll be responsible for triggering discharge? Do the same ions that ellicit discharge of nematocysts isolated from the anemone cause discharge in those from the nudibranch, or have they been modified? My experiments intended approach each of these questions. Materials and Methods Collection and care of animals Anthopleura elegantissima were obtained from tide pools at Hopkins Marine Station, Pacific Grove, CA; Aeolidea papillosa and Metridium senile were collected at a depth of about 40 feet from pilings of the Monterey Vharf. Animals were maintained in tanks through which fresh sea water flowed at 15-18 C. A. papillosa fed on A. elegantissima placed in the tanks for that purpose Isolation of nematocysts by homogenization Scissors were used to excise cerata from A. papillosa in sea water. The cerata were then blotted gently on paper towels to remove excess mucus and rinsed several times in the medium in which they would be homogenized. Homogenization media used were filtered sea water, intracellular medium, and 1% saponin in distilled water. Intracellular medium contained (in mM): K-Gluconate, 225: Mannitol, 185: Glycine, 300; Nacl, 20: MgCl,.5. HEPES, 20; EGTA, 2: NahC03. 2; all this adjusted to pH 7.2. Excised cerata where then placed in a Dounce homogenizer and the chosen homogenization medium added at a liquid to tissue ratio of about 10:1. After gentle homogenization and a five minute incubation, homogenate was divided into separate aliquots and centrifuged two minutes at 840 g to condense the isolated cnidae. One of the aliquots containing only homogenization medium plus tissue debris was set aside as a control. Additional aliquots were subjected to two experimental conditions: (1) An ion was added to the supernatent or (2) supernatant was removed and test medium added. In the first case, all soluble cellular components remained in the aliquot in the second, cnidae were subjected to pure ionic conditions. Because the chelating action of EGTA produces four protons per Ca2 Ca Cl, added to homogenate in intracellular medium required pH adjustment. To achieve a net addition of 2mMCa to the homogenate, I added 4mM CaCl,, expecting the EGTA to absorb 2mM, then adjusted the pH with .IM KOH prior to incubation. After a five minute incubation period, aliquots were stained with methylene blue and inspected under the microscope (200X). Numbers of discharged and undischarged cnidae were recorded on a manual counter and appear in Table 1 Behavior of nematocysts in vivo Intact cerata excised and blotted as described above were rinsed in each of the following media: 05M KCI, Cacl,. Nacl, and MgCl, 0.SM KCI, Cacl, Nacl, and MgCl, sea water intracellular medium: 1% saponin; and one of several concentrations of sodium citrate. The ceras was then placed on a slide in the rinse medium plus one drop of 1% methylene blue and a cover slip placed over it. Liquid could be gradually blotted from the edge of the cover slip until the pressure produced caused the ceras to expel nematocysts. The percentage of nematocysts that discharged could then be counted. Isolation of intact enidophages Placing the ceras on a slide and cutting it lengthwise with a razor blade I could expose the white cnidosac and tease it free of the surrounding tissues. The cnidosac could then be minced in sea water using razor blade and forceps to free intact cnidophage cells visible at 200X under the phase microscope. A water immersion lens was used so the preparation could be viewed without a cover slip. By this technique, both nematocysts and cnidophages could be obtained in solution, 100% undischarged. Solutions containing various second messenger inducers could then be pipetted directly onto the slide and their effect on both cnidophages and nematocysts observed. (For chemicals and concentrations used, see table 3). Having watched the cnidophages as I added an inducer, I then incubated the slide 10-20 minutes at 15 C and again counted the number of intact cnidophages. As a control, intact cerata were forced to extrude nematocysts into media containing the same concentration of each inducer to be sure that the cnidae still discharged normally. Results Nematocysts Isolated by Homogenization Data from homogenization experiments appears in Table 1. Under no condition could nematocysts isolated by this method be induced to show a significant frequency of discharge. Spirocysts isolated in intracellular medium, on the other hand, discharged readily upon exposure to seawater. Nematocyst discharge in vivo In Aeolidea papillosa I observed five to twelve nematocysts bundled parallel to one another in each cnidophage (Figure 2). The cnidophages seem to be maintained at no particular orientation within the cnidosac (although Greenwood and Mariscal observed otherwise for a different species, 1984). When a ceras placed under a cover slip experiences pressure, a pore opens at its tip and muscular pulsing forces the cnidophages through it one at a time. In hundreds of observations, tubules were never seen to evert within the cnidosac or cnidopore but a large percentage did so immediately upon emerging from the pore. Whether the cnidophage membrane ruptures before or after its resident nematocysts discharge is difficult to determine. As more cnidophages emerge from the pore, they accumulate in a tangled mass. (Figure 3) Table 2 shows the percent discharge for nematocysts emerging in vivo into different ionic media. For cerata from the same animal, this assay should test the effect of certain ions on nematocysts that have experienced similar conditions during extrusion. In calcium-free media, including Na-citrate, intracellular medium with EGTA, and MgCl, the cerata ceased contracting, but extrusion could be forced using pressure on the cover slip. Agents eliciting second messenger response Data from the second messenger experiments appear in Table 3. Isolated cnidophages could be observed under the microscope while the second-messenger-inducing solution was added; this observation yields the number in the second column. Discharge after a 10 to 20 minute incubation appears in the third column. Positive percentages indicate that after in cubation, some cnidophages were observed with everted tubules protruding through their membranes. Discussion Although previous researchers have assumed that contact with sea water triggers eversion of the tubule for nematocysts extruded from Aeolidcerata (Greenwood and Mariscal, 1984), the balance of my observations suggest otherwise. Something in the pulsing action of the cerata and perhaps passage through the cnidopore seems critical for efficient discharge. Hidaka and Mariscal (1988) note that nematocysts isolated in different media from the sea anemone Calliactis tricolor show differential tendencies to discharge in response to the same stimulus. The procedures I used suggest that nematocysts stored by the nudibranch also fail to discharge normally in isolation: Nematocysts isolated by homogenization could not be induced to discharge even under the most extreme conditions (.17 saponin, triton, or SDS in ddH,0). Digestion of cerata with collagenase in sea water yielded a suspension of free nematocysts in which less than one percent had discharged. Furthermore, cnidophages and free nematocysts could be released into sea water by dissection of the cnidosac without eliciting discharge. Cerata from the same animal, placed under a cover slip, extruded clumps of nematocysts that showed 90% discharge, These results indicate that contractile action of the cerata or passage through the cnidopore must play some essential role in activating the nematocysts. None of the ions tested seems essential for triggering discharge from intact cerata (Table 2). Differences in 7 discharge are deceptive because the 0.8 and U.5M cases were measured using cerata from different animals. Presumably, such variables as the health of the animal and the age of its nematocysts influence the viability of its defensive system. The observation that divalent cations appear to stabalize undischarged nematocysts from sea anemones (Blanquet, 1970) seems to hold for Aolidea only in the case of high concentrations of Mg Ca2 on the other hand, seems to slightly enhance discharge. As the cerata are placed under a cover slip, they continue contracting. Several minutes of contraction and extension may be necessary before cnidophages begin passing through the cnidopore. In Ca-free media, which prevents natural pulsating of the cerata, discharge is consistently reduced. The solutions in which cerata cease contracting are MgCl Intracellular Medium-EGTA, and Sodium Citrate. Under these conditions, cerata must be forced to extrude nematocysts using pressure on the cover slip The nematocysts emerge in abnormally large masses and few threads evert. Passage through the cnidopore, therefore, is not sufficient to induce discharge Mge seems to be a secondary effector in the case of nematocyst discharge; its critical action is on the activity of the cerata. The hypothesis that external calcium stabalizes undischarged capsules becomes more suspect considering the action of sodium citrate. My data are exactly in accord with Blanquet's (1970) for anemones that discharge increases as Na-citrate concentration declines. (Figure 4) This is the opposite of what one would expect because citrate, as a calcium chelator, should reduce the level of calcium in the medium as its osmolarity rises. Discharge should therefore become more frequent at high concentrations of citrate. I would suggest that the effect of 1.0 M citrate results from its viscosity rather than its ionic effects a viscous solution may discourage eversion of the tubule This could be tested by substituting a sodium citrate concentration of comparable Ca2 chelating action for EGTA in intracellular medium and comparing the rate of discharge for inact cerata. The failure of all kinds of agents to elicit nematocyst discharge in vitro leads to speculation that the cnidophage cells play a role in triggering eversion of the thread. One possible mechanism is that contact of the cnidophage membrane with sea vater activates a second messenger within the cell that then activates specialized proteins, signalling its nematocysts to fire. Another possibility is that some helper cell sends a signal to the cnidophage that then activates a second messenger. The second messenger hypothesis appears reasonable especially because of the recent discovery that anemone's have receptor-bearing cells next to the cnidocytes containing nematocysts (Mariscal, Conklin, and Bigger. 1978). Upon sensing food material, the effector cell is thought to signal the adjacent cell to fire its nematocysts. Application of five second-messenger stimulating agents to intact cnidophages, however, failed to elicit a response. As nematocysts expelled by an intact cerata into each of these agents discharged normally, the agent could not have poisoned them to discharge. One would therefore have to conclude that none of the second messengers tested is work here. Some incidental observations about the physiology of isolated cnidophages raises additional questions. The cnidophage lumen is almost completely occupied by the nematocysts it contains. Usually two large nematocysts dictate its long dimension and a number of smaller ones cluster parallel to these at one end. (Figure 2) Five or more flagella may be observed at one end of the cell, which beat frantically in sea water. In the cnidosac, cnidophages are densely packed with little opportunity for motion. Under close inspection, the contents of the semi-transparent cnidosac do not seem to be moving around. One might inquire, then, why are the cnidophages flagellated They seem to have no need of locomotion within the cnidosac and usually do not survive extrusion from it. Perhaps the flagella are an evolutionary remnant in an organism that was once independently viable. They also may facilitate communication among cnidophages or between cnidophages and other cells. It would be interesting to make a CDNA library from the cnidophage RNA and screen it against the nudibranch genome to asses their similarity or difference. Another interesting question is to what extent cnidophage cells take up 10 anemone parts. Of course they acquire nematocysts, but might they also take up cnidocyte receptors and other proteins critical for nematocyst discharge as well? The extent of their scavenging remains to be shown. Summary While I have not succeeded in establishing how nematocysts work,I have ruled out some possibilities. From the homogenization, digestion, and dissection results, one can surmise that an interaction with the surrounding tissues is necessary for efficient discharge. However, nematocysts within theis cnidophages do not respond to any of the second messengers examined. The development of procedures for isolating intact cnidophages allowed me to examine their morphology more closely, raising interesting questions as to their evolutionary origin. Literature Cited Anderson, P. and Mckay, C. 1987. "The electrophysiology of cnidocytes.“ J. Exp. Biol., 133,215-230. Blanquet, R. 1970. Ionic effects on discharge of the isolated and in situnematocysts of the sea anemone, Aiptasia pallida A possible role of calcium." Comp. Biochem. Physiol, 35. 451-461. Campbell, R.D. 1987. "Organization of the nematocyst battery in the tentacle of hydra: Arrangement of the complex anchoring junctions between nematocytes, epithelial cells, and basement membrane." Cell Tissue Res., 249, 647-655. Cargo, K.G. and Burnett, J.W. 1982. "Observations on the ultrastructure and defensive behavior of the cnidosac of Cratena pilata," Veliger 24,325-327. Conklin, E.J. and Mariscal, R.N. 1977. Feeding behaviour, ceras structure, and nematocyst storage in the aeolid nudibranch Spurilla neapolitana,"Bull Mar Sci 27. 658-667. Greenwood, PG. and Mariscal, R.N. 1984. "Immature nematocyst incorporation by the aeolid nudibranch Spurilla neapolitana, "Mar. Biol. 80.35-38. Greenwood, P.G. and Mariscal, R.N. 1984. The utilization of cnidarian nematocysts by the aeolid nidibranchs.“ Tissue and Cell 16,719-730. Hidika, M. and Mariscal, R.N. 1988. Effects of ions on nematocysts isolated from acontia of the sea anemone Calliactis tricolorby different methods.“ J. Exp. Biol, 136.23-34. Holstein. T. and Tardent, P. 1984. "An ultrahigh-speed analysis of exocytosis: Nematocyst discharge. Science 223,830-832. Kalker, H. and Schmekel, L. 1976. Structure and function of the cnidosac of the aeolidoidea," Zoomorphology 86. 41-60. Lentz, T.L. and Barrnett, R.J. 1962. The effect of enzyme substrates and pharmacological agents on nematocyst discharge. J Exp. 2ool, 149,33-38. Lubbock, R. and Amos, W.B. 1981. "Removal of bound calcium from nematocyst contents causes discharge.“ Nature 290. 500-501. Lubboch, R., Gupta, B., and Hall, T. 1981. "Novel role of calcium in exocytosis: Mechanism of nematocyst discharge as shown by x-ray microanalysis.“ Proc. Natl. Acad. Sci. 78, no. 6. 3624-3628. 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Table 1: Discharge of Nematocysts Isolated by Homogenization Homogenization lons % Discharge Medium Added Metridium Elegantissima Aolidea Sea Water 1% Saponin in distilled H,0 Intracellular Medium lonic media added to supernatent, yeilding net conc. 90 spirocysts. 2 mM Cacl, 0 nematocysts 6 mM Cacl2 12 mM Cacl2 0.5 5M KCI Homogenized in intracellular med., supernatant decanted and replaced with: Sea Water 1% Saponin in DW 14 Table 2: Discharge of Nematocysts Iszuing from Intact Cerata Medium 7 discharge (number counted) 05MKCI 5 (100) 0.5 MNaci 5 (100) 0.5 M Cac12 7 (100) 0.5 MMgCI, 20 (25) 25 (75) 0.8 M Caci, 74(35) 0.8 MNaci 67 (33) 0.8M KCI 90 (100) 0.8 MMgCI, 3 (100) 15 (100) Intracellular Medium 40 (100) 17 Saponin in ddH,0 95 (100) Na Citrate 0.75 M 10 (50) 05 M 20 (50) 0.25 40 (65) Table 3: Nematocyst Discharge from Cnidophages in Response to Second Messengers Intracellular Immediate 7 discharge after Agent Added discharge 10-20 minute incubation effect stimulates protein TPA 02 uM kinase C Ca lonophore release of stored calcium 5uM stimulates adenylate Forskolin cyclase; resultin 100 uM rise in levels of cAMP activates protein kinase A 10 rise in NH.CI 20 mM in SW, intracellular ph pH 9.0 Na-Acetate decrease in intracellular ph 20mM in SW. pH 6.0 Figure Legends Figure 1: Anatomy of the aeolid nudibranch. CA, cerata. CN, cnidophage. A. anus. From Kalker and Schmekel. 1976. Figure 2: Cnidophage cell containing nematocysts. Figure 3: Appearance of nematocyst discharge from a ceras tip. Figure 4: Variation in discharge in different concentrations of sodium citrate. W = Figure 1 N00 RH Figure 2 Gf el tf Figure 3 L C 0 Z L O I I O 0 V 8 1 1 -C O 1 N ebipusigsoouen