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
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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.
Mariscal, R.N. et al. 1979. The putative sensory receptors associated with the cnidae of
cnidarians." Scanning Electron Microscopy 11. 959-966.
Mckay, M. C. and Anderson. PA. 1988. Preparation and properties of cnidocytes from
the sea anemone Anthopleura elegantissima. Biol. Bull. 174,47-53.
Pantin, C.F.A. 1942. The cnidoblasts of actinians.“ J. Exp. Zool, 39,294-310.
Watson, G.M., and Mariscal, R.N. "Ultrastructure of nematocyst discharge in tentacles of
the sea anemone Haliplanella luciæ. "Tissue and Cell 17,199-213.
Weber, J., Klug. M., and Tardent, P. 1987. "Some physical and chemical properties of
purified nematocysts of Hydra attenuata."Comp. Biochem Phsiol, 88B. 885-862.
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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.
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