Coelenterate Cnidae Capsules: Disulfide Linkages Revealed by Silver Cytochemistry and Their Differential Responses to Thiol Reagents

16 Pages·1997·6.9 MB·English

by  W M Goldberg

Reference: Bid. Bull 192: 1-16.(February, 1997)
Coelenterate Cnidae Capsules: Bisulfide Linkages
Revealed by Silver Cytochemistry and Their
Differential Responses to Thiol Reagents
WALTER M. GOLDBERG AND GEORGE T. TAYLOR
Electron MicroscopyLaboratory, DepartmentofBiologicalSciences. Florida International
University. UniversityPark. Miami. Florida 33199
Abstract. The sulfurcytochemistry ofcnidae from the P. physalis discharged and appeared to dissolve rapidly
Portuguese man-of-war Physalia physalis. the scypho- in the presence ofthis reagent, whereas small isorhizas
zoan Cassiopeiaxamachana. and the black coral Cirrhi- frombothP.physalisandC.xamachanadischarged,but
pathesluetkeniwasevaluatedonthebasisofelectron mi- dissolved slowly ifat all. The discharge and solution re-
croscopy. X-ray microanalysis, aminoacid analysis, and sponses ofthe capsule coincided with the complete de-
response to disulfide reducingagents. The cnidae exam- velopment ofthe tubule. Cnidae containingan undevel-
ined included large and small holotrichous isorhizas in oped or partially developed tubule were resistant to
P.physalis, anothersmall isorhizain C. xamachana. and DTT. displayed aweak capsularargentophilia, and con-
both spirocysts and microbasic mastigophore nemato- tained background levels ofsulfur; these results suggest
cysts in C. leutkeni. A strong reaction with methena- that formation of disulfide linkages is one of the final
mine-silver reagent was characteristic ofall cnidae cap- steps in capsular maturation. In contrast, mature nema-
sules,butthepatternandextentofthatargentophiliawas tocystandspirocystcapsulesinC. leutkenitentacleswere
dependent upon the type ofcnida and its state ofmatu- resistant to DTTamongother reagents, despitethe pres-
rity. The large isorhizas ofP. physalis reacted primarily ence ofdisulfides. Thissuggests that othertypes ofcova-
in the outermost capsule layers, but in C. xamachana lent,intermolecularlinkagescouldplayaprominentrole
isorhizas,silverstainedtheentirecapsulewith theexcep- in thedevelopment ofcapsularstability in thisspecies.
tion ofthe outermost region. The small isorhizas ofP.
physalis and the mastigophore capsules of C. leutkeni Introduction
stained throughout, whereas the spirocyst capsules were
outlined bysilver,clearlydelineatingthe innerandouter Coelenterate cnidae are among the most complex in-
layers. All ofthese reactions were abolished with alkyl- tracellular secretion products known (Gupta and Hall,
ation, but only after treatment with disulfide reducing 1984). Each iscomposedofadouble-walledcapsulecon-
agents; alkylation alone diminished silver staining only taining an inverted tubule. During eversion the tubule
slightly, indicating that the argentophilic response was discharges explosively, completing the process within 3
due primarily to disulfide linkages. The cystine content ms, one ofthe fastest mechanical events known in the
ofthese cnidae varied from 4.1 to 4.7 mole percent fora biological sciences (Holstein and Tardent, 1984). More
given species, but amino acid analyses did not separate than 30 types ofcnidae have been described, and they
componentsofthecnidom. are classified into three groups: nematocysts. spirocysts,
Cnidae, both within and among species, exhibited and ptychocysts (e.g., Mariscal, 1974, 1984). Hydrozo-
differential responses to the disulfide reducing agent di- anshavethegreatestvarietyofnematocysts,andscypho-
thiothreitol (DTT). Isolated, unfixed, large isorhizas of zoans have the least, but only anthozoans produce all
three types. Ptychocysts occur only in cerianthid anem-
ones and are the most phylogenetically restricted ofthe
Received IVJuly 1996;accepted 16October 1996. cnidae. Spirocysts are also limited in their distribution.
W. M. GOLDBERG AND G.T. TAYLOR
occurring only in the tentacles of various anthozoans; from various localities in southeast Florida; Cassiopeia
but spirocysts are also quite common, often outnum- \iinuichuna Bigelow (Scyphozoa: Rhizostomae) from
bering tentacular nematocysts (Mariscal and McLean, several nearshore locationsin the Florida Keys, and Cir-
1976;GoldbergandTaylor' 1989). rhipathesluetkeniBrook(Anthozoa: Antipatharia) from
m
Nematocystsin particularhavebeenexamined closely, adepth of25 offHollywood, Florida.
yielding details of structure and function relationships. Nematocystbatteriesseparated from fixedtentaclesof
Forexample, upon discharge, thecapsule mustwithstand P.physalis, individual tentaclesisolatedfrom C. luetkeni
internal pressures ofup to 140 bar duringeversion (Lub- polyps,and oral vesicles thebaglike,ovalstructuresas-
bock and Amos, 1981; Tardent, 1988). a feat requiring sociated with the oral arms (see Bigelow, 1900) from
enormous tensile strength. Recent work has shown that C. xanuic/iana were dissected using iridectomy scissors
disulride-linked, woven mini-collagenscomposingthe in- under low-power microscopy. All of these cnidae-con-
ternal wall ofthe nematocyst capsule (Kurz el at., 1991) taining structures, referred to as tentacles or tentacular
may be the key structural element in the resistance of tissues, were prepared for transmission electron micros-
these cnidae to such pressures. The tensile strength de- copy (TEM) by fixation at room temperature for 2-4 h
rived from thiscapsule structure isestimated to be nearly in an artificial seawatersolution containing2.5% glutar-
ashigh asthatofsteel (Holstein eta/., 1994). aldehyde and 1.0% paraformaldehyde in 0.1 v\/cacodyMl-
The occurrence ofdisulfide-linked collagens in nema- ate bufferat pH 8.0. The tissue wasthen stored in0.1
tocysts was first established by Blanquet and Lenhoff cacodylate buffer at 4C. Post-fixation with osmium is
(1966) after the suggestion by Brown (1950), and subse- incompatiblewith silverstaining(e.g.. Hayat, 1993) and
quently by Yanagita and Wada (1954), that since coelen- was omitted. Tentacular tissues were dehydrated
teratecnidaearesolublein disulfide reducingagents, they through ethanols and embedded in Spurr resin. Thick
mightbecomposed ofkeratins. Hamon(1955(confirmed sections examined in the light microscope were con-
the earlier observations ofdisulfide linkages by demon- trasted with 0.1%. toluidine blue in 1% borax. For TEM
stratingthepresenceofcystinehistochemically. However, we used a Philips EM 300 electron microscope operated
Blanquet and LenhofYclearly showed that cystine is re- at 60 kv. No contrast agents other than silver were em-
sponsible for stabilizing collagenous proteins, the domi- ployed in transmission microscopy.
nant components of the nematocyst capsule. They also Cnidae fixedasabovewerealsoexaminedbyscanning
showed that the cnidae at their disposal dissolved in a electron microscopy (SEM). Aldehyde fixation was fol-
M
numberofdisulfide reducingagents includingdithiothre- lowedbyosmication using 1%. OsO4 in 0.1 cacodylate
itol. sodium thioglycolate. and mercaptoethanol. buffer, pH 8.0, for 1 h at room temperature. Dehydra-
Disulfide linkages have since been shown to be wide- tion in ethanols was followed by critical point drying
spread in nematocyst capsulesjudging from amino acid (CPD) with CO: asthe transitional fluid, or by cryofrac-
composition (Fishman and Levy, 1967; Blanquet, 1988; ture from 100; ethanol in liquid nitrogen priorto CPD.
Brand el at.. 1993) and X-ray microanalysis revealingthe Tentacles were then sputter-coated with Au-Pd and ex-
presenceofsulfur(Mariscal. 1980, 1984. 1988). However, amined inan ISI Super3Ascanningelectron microscope
althoughthenematocystsfromavarietyofcnidariansdis- operated at 10 or 15 kv.
solvequickly in disulfide reducingagentsas might bepre-
dicted, some nematocysts and spirocystsappear to be re- Silverstainingfordisulfidegroups
sistant to such treatment (Mariscal and Lenhoff, 1969; The methenamine-silver stain (Rambourg 1967;
Mariscal. 1971). Despite a number ofsubsequent chemi- Locke and Krishnan, 1971) was employed for general
cal studies (see review by Blanquet. 1988; Brand el a/., electron contrast and sulfur cytochemistry. All reagents
1993). little progress has been made in answering the were made fresh daily using double-distilled, deionized
questionsraisedby Mariscal and LenhofTsoriginal obser- (ddd) water, and all steps employed constant agitation
vations. To this end, we have examined the chemistry of onan orbital rotator. Fixedtentaculartissueswererinsed
cnidaecapsulesinthreecoelenterates.each representinga several timesin ddd-water, then treated with 5% sodium
classwithinthephylum. Thesulfurcytochemistryofeach metabisulfite for 10 min to block pre-existing aldehyde
type of cnida is confirmed by X-ray microanalysis and groupsorthoseintroduced bythefixative. Afterwashing
correlated with amino acid composition, degree ofmatu- in three additional changes of ddd-water, tissues were
rity, and responsetodisulfide reducingagents. immersed in methenamine-silver reagent and placed in
closed 1.5-ml polypropylene microcentrifuge tubes in a
Materialsand Methods
70C oven for 30 min. After a briefddd-water rinse, all
Microscopy tissues were treated with 5% sodium thiosulfate to re-
Three species were collected for this study, including move unreduced silver deposits, then rinsed with ddd-
Physalia physalis Lamarck (Hydrozoa: Siphonophora) wateragain beforedehydration and embedment.
DISULFIDES IN CNIDAE CAPSULES
Cytochemicalcontrolpr showing maximum, minimum, and mean net counts
wasconstructed foreach typeofcnida
Beforesilverstaining, additional aldehyde-fixed tenta-
clesweretreatedwith thedisulfidereducingagentdithio- Isolation olcnidaeandaminoacidanalysis
M
tphhroesitpohlat(e=bDuTffTe,rpCHlel8.a0nda'ts3r7eCagefnotr)900.m2inA.l Sionm0e.0s5am- oraFlrevesshilcylecso,lalencdtewdhPo.lephsyesgamliesnttsenotfacClesl,ueCt.kxenaimcaoclhonaineas
4C
ples were stained with methenamine-silver and exam- wereallowedtoautolyzeinseawaterat overaperiod
ined without further treatment; otherswere treated with of2-7 days. Debris was removed by filtration through a
iodoaceticacidtoalkylate naturallyoccurringsulfhydryl coarse nylon mesh, and the filtrate was treated briefly
groups as well as those produced by disulfide reduction with a sonicator probe to disperse the remaining ma-
(alkylation blocMkade). Iodoacetic acid (0.1 Af) was pre- terial. Recovery of undischarged cnidae from P.
pared with 0.2 boric acid in 50% jV-propanol accord- physalis required an initial low-speed centrifugation
ingtothemethodofSwift( 1968). Becausewe usedtissue (200 X g) for 4 h over concentrated sucrose (approxi-
rather than sections, the reaction time for this blockade mately 2.5 Af). The sucrose layerwasdiluted and cnidae
was extended from 4 h at room temperature to 18h. were concentrated by centMrifugation, then resuspended
Blockadedtissueswererinsed firstwith 20%7V-propanol, in a small volume of0.1 cacodylate buffer, pH 8.M0.
then several times with ddd-water, and treated with me- The crude, buffered autolysate was layered over 2.0
thenamine-silver as above. Samples treated with io- sucrose and centrifuged at 10 X g for an additional
doacetic acid alone prepared as above, or with the boric 60 min, or allowed to settle at 4 overnight. A clean ne-
acid-propanol solvent alone, were also examined both matocyst preparation was obtained after repeating this
withand without silvertreatment, asweretissuestreated process. The filtered C. xamachanaautolysate wassepa-
with DTTalone. Parallelexperimentsemployedalkaline rated from debris by centrifigation into 1.25 AI sucrose
M
0.3 thioglycolic acid prepared and used under the for 1 hat 1000 X g. Cnidaein thesucroselayerwerecon-
same conditions as DTT for disulfide reduction, and centrated, resuspended in cacodylate buffer, and briefly
0.1 MTV-ethyl maleimidepreparedaccordingto Kiernan sonicated after addition of 1 yul ml'1 Triton X-100. The
(1990) to block sulfhydryl groups. These sequential con- crudecnidae were filtered through a 10-/um nylon mesh,
trol treatments (disulfide reduction followed by alkyl layered onto a discontinuousgradient of 1.75, 2.0, 2.25,
bockade)will be referred toaspost-reduction alkylation. and 2.5 Af sucrose, and spun for 1 h with a swinging
buckMet rotor at 2000 X g. Cnidae were in the 2.25 and
2.5 layers. This process was repeated 2-3 times. The
Elementalcomposition C. luetkeni autolysate contained a considerable amount
Sections about 100-nm thick (dark gold interference of mucus and was treated with 0.5%- cetylpyridinium
color)fromtissuestreatedwith methenamine-silverwere chloride in addition toTriton X-100. Thetreatmentand
mounted on carbon-coated copper grids and examined procedurewasotherwisethesameas forC. xamachana.
in a Philips EM 300 transmission electron microscope Clean, isolated cnidae from each species were taken
operating at 100 kv. X-ray microanalysis employed a from buffer to distilled water and disrupted with a soni-
Link Analytical EDS system. Thegoniometerwas tilted catorprobe. The fragments were recovered by briefcen-
at 36 and counts were obtained using a spot size of trifugation, rinsed with distilled water, then dehydrated
250 nm over an interval of600 s real time. Count rates in ethanols and oven-dried. Cnidae fragments yielding
wNeorefe1w8e0r0t-h2a5n0f0oucrpsu;nddiesacdhtairmgeedvacrniieddaefroofmea2c0h%tytpoe2(5d%e.- f1o-r23n0&moifnprtootceoinnvewretrecyosxtiidnieziendtoincy9s5te%icpaecrifdoramnidcwaecried
csm/cairtaionbcae,ydsatbnsedlforbwoo)mtwhPe.raepnhseycmsaaanltnioescdyt,sentitnaaccnlleud,diaonngseptifrworocoytmsytpCe.fsrxooafmmnaCe-.- AsHuCmb1isneqofuoerancti1ld8ysmhiwynedrroeulnyddzeeerrdivbiatynizmeiadctrmwooiwstahpvheeErddemigaeonsf'tsinoinrteirnaogg6eennT.tV
luctkeni. In all but C. luetkeni. thespot wasmoved every (phenylisothiocyanate see Heinrickson and Meredith,
s5u0l-e.60Instthoeanlaottthererspleocciaetsi,onthoenspthoetswaamsemnoevmeadtoecvyesrtyc3a0p-- m1a98n4c)ealniqduidqucahntriotmaatteodgrbayphryevaetrsFel-oprhiadsaeSthaitgeh-Upneirvfeorr--
ts5ym0palselaonsfidzcenciohdfaatnhgienecdCa.ptsoluulacetsnk.eeTwnihcuwnsaisdeaaacehcvoe6mr0py0o-s1si5ct0oeusnodtfufefoorutroeantcehh-e slfieotnrygmtAehndaolifynt2it5cr4ailplnimLc.aatbeo,Arnawatilotyrhsieetssh,eofucsneiiandcgaheasopdfeecteieaeccsthiwoernerpewliapcveaert--e
matocysts and four spirocysts; four such 600-s counts representinga single individual orcolony.
were taken from a total of 16 cnidae in this species. X- EffectsofDTT. thioglycolicacid, andcollagenase
ray spectra of Spurr resin (blank sections) served as a Isolated cnidaestored in 0.1 Afcacodylatebufferwere
means of determining background. A composite peak taken todistilledwater. Theeffectsofdisulfidereduction
W. M. GOLDBERG AND G.T. TAYLOR
Q'm&
-.
,
^^i.'^:m^^^w^^
,- --f>^^<:
,' --- T^m^ii'^
Figures 1-7. Cnidaefrom Physaliaphysalis.
D1SULFIDES IN CN1DAE CAPSULES
wereexaminedfor 15 minafterplacinga 10-/uldropcon- condition. Thedesignation ofthesenematocystsasholo-
taining several hundred cniMdae on a slide, followed bMy trichous isorhizas seems appropriate and is consistent
25 ftl of0.2 A/ DTT or 0.3 thioglycolic acid in 0.2 with identifications made previously (Mariscal, 1974;
bicarbonatebuffer, pH 10.3 (Brand etui.. 1993). Isolated Brand et ai. 1993).
cnidae were also tested with collagenase (Type 1A: Cnidae from P. physalistissuestreated with methena-
320 units mg"1 and 1.7 units mixed protease, Sigma mine-silverdemonstrated a clear and consistent pattern
Chemical Co., St. Louis, MO)byexposingthem to 1500 ofargentophilia. Thelargeisorhizaswerealwaysstrongly
enzyme units in TES buffer, pH 7.5 (10:1 enzxnidae silver-positive, but only in the outermost portion ofthe
vol), for 5 h at 37C. Cnidae were recovered by centrifu- capsule (Fig. 4). Deeper regions, delineated by what ap-
gation. washed in distilled water, and again exposed to peared to be fibrous annuli, were only lightly stained.
DTT orthioglycolicacid asabove. These electron-opaque annuli subdivided the capsule
intoas many asfivedistinct layers. In contrast, thesmall
Results isorhiza capsules were most often completely blackened
with metallic silver (Figs. 4, 5). In less intensely stained
Isolatedcnidaeandelectron microscopy capsules, one or two electron-opaque annuli could be
Physaliaphysalis. Clean, largely undischarged nema- distinguished, but in small isorhizas these layers did not
tocyst preparationswere obtained from P. physalis (Fig. react differentially with silver. The annuli were present
1). Cnidae isolated in sucrose were remarkably difficult in isorhizas that were untreated except for aldehyde fix-
to discharge and, in contrast to results reported by Lane ation, andweretherefore unrelated tothesilverstain.
(1960), were often unresponsive to centrifugation or to In P. physalis and all other species examined, the
mM
immersion in distilled water, 50 sodium citrate, N- background stain varied in intensity, probably due in
HC1 or A'-NaOH. despite capsular permeability to tolu- part to variations in temperature during the 30-min in-
idine blue. Twosizeclasses(diameters 8-12 /urn and 20- terval at 70C. Figure 4 shows a low-background prepa-
35 //m) were obtained from tentacles (Fig. 2). These ration inwhich somegeneralized stainingofmembranes
ranges are in general agreement with the measurements is evident. However, nothing in the tissue compares to
obtained by Laneand Dodge(1958), Hulet et at. (1974), the intensity ofthe capsule stain. Figure 5 shows a small
andCormierand Hessinger(1980). The largerofthetwo isorhiza with typically strong capsular argentophilia but
wascharacterizedbya uniform tubulediameterofabout with higher background staining of membranes and
2.5Mm. with spines of about equal size (0.9-1.2 ^m), small vesicles. Nuclei and mucus cell secretory inclu-
uniformly distributed alongits length in three rows(Fig. sionswerealsostained. Thenematocysttubulewasquite
3).Thesmallercnidaecontainedsimilartubulesandspi- variably electron-opaque (cf. Figs. 4 and 5), and silver
nation. but these were not examined in the discharged depositswereoftenobservedonthetubuleperiphery. Al-
Figure 1. Large and small isorhizas isolated from tentacular tissue in sucrose. Darkheld microscopy.
Scalebar= 100^m.
Figure2. Freeze-fractureSEMofnematocystbatterycross-sectionshowinglargeisorhizaandsurround-
ingcnidocyte(ct)flankedbytwosmallisorhizas.Theopercula(arrows)arevisibleatthetopofeach;battery
surfaceisattheupperleft.Scalebar= 5^m.
Figure3. Evertedtubuleoflargeisorhizashowingarrangementofthreespiral rowsofspines.Scalebar
= 5Mm.
Figure4. Low-magnificationoverviewofmethenamine-silverpreparationshowingtwosmallisorhizas
(topandright)withsurroundingtissue,andasectionofalargeisorhiza(left).Notethatsmallcnidaecapsules
arecompletelyinvestedwithelectron-opaquesilverdeposits,whereasthelargeisorhizaisprimarilyreactive
in the outer capsule: annuli marking inner capsule layers are noted by arrows. The tubule (t) ofsmall
isorhizasdisplayslittlereactivitytowardsilver.Scalebar= 2^m.
Figure5. Detailofmethenamine-silverreactiononsmallisorhizaandsurroundingcnidocyte.Thecap-
sule isclearly delineated from surrounding cellular material by the degree ofargentophilia. A tangential
sectionthroughthecnidocilcomplex(cc)isshownattheupperleft. Extracellularvesicles(upperright)are
silver-positive,asismucussecretory material(cleararrow).Tissuewastreatedwithiodoaceticacid,butnot
withdisulfidereducingagent.Scalebar= 1 ^m.
Figure6. Low-magnification overviewoftentaculartissueafterDTT-iodoaceticacidblockadeofme-
thcnaminesilverreaction.Notethatnucleus(n),numerouscnidocytevesicles(arrows),andmembranesare
reactive,butthecapsular(c)responseisallbutcompletelyblockedbyalkylation.Scalebar= 2^m.
Figure7. Detailofblockadeonsmallisorhiza.Notethattheoperculum(op),theperipheryofthetubule
(solid, curved arrows), and thecnidocyte cytoplasm are reactive, but thecapsule is not. Electron-opaque
spinesvisibleinthetubulecenter(clear,curvedarrows)arenotargentophilic.Scalebar= 1 /jm.
W. M. GOLDBERG AND G.T. TAYLOR
though the opacity ofthe spines in the center ofthe tu- occurring electron-opaque material in the tubule
bule could be confused with silverstain, they were pres- center.
ent in unstained controls (see also micrographs in Hulet Mature isorhizas were often closely accompanied by
el a/., 1974, and Hessinger and Ford, 1988) and were various stages oftheirdevelopment. The most common
therefore unrelated toargentophilia. of these were capsules containing an electron-opaque
ControlproceduresusingDTTorthioglycolicacidfol- matrix in which the tubule had not yet developed, or in
lowed by iodoacetate or ethyl maleimide alkylation es- which an incipient tubule could be observed (Fig. 10).
sentially eliminated capsular argentophilia, while back- Werefertothe formerasimmaturecnidaeandthe latter
ground staining remained on nuclei, cnidocyte mem- as submature. In both cases, the capsule was substan-
branes, and many extracapsular vesicles (Fig. 6). At tially lessargentophilic than the mature cnidae. The im-
highermagnificationsomenonspecificsilverwasstillvis- mature stages reacted variably with silver; reactions
iblearoundthetubuleperiphery, andtheoperculum was ranged from diffuse and nonlocalized deposits to no re-
alsostained (Fig. 7), suggestingthat these structures may action atall. The submature cnidae also exhibited varia-
have a different chemical composition from that ofthe tion incapsularargentophilia, which rangedfromdiffuse
capsule. Tissues treated with DTT or thioglycolic acid deposits to more concentrated metallic silver, but was
alone exhibited a somewhat less intense capsular argen- always less than in the mature cnidae. In addition to ac-
tophiliaandahigherbackground,butwereotherwisenot quiringargentophiliaduring the transition from subma-
significantly different from untreated tissues. Treatment ture to the mature condition, the capsule exhibited a
with disulfide reducing agents alone, however, removed 20%-50% decrease in thickness. Moreover, theelectron-
the electron-opaque annuli that subdivided the capsule, lucent tubule ofsubmature cnidae and theircontrasting
resulting in the uniformly electron-lucent appearance of electron-opaquematrix(Figs. 10, 11 inset)changedtoan
capsules in Figures 6 and 7. Control tissues treated with electron-opaque tubule in an electron-gray matrix dur-
propanol and boric acid, or iodoacetic acid (as in Fig. ing maturation. The latter change was the most consis-
4),orethyl maleimidealonewereindistinguishable from tent indicator ofcapsules most likely to exhibit a strong
experimental groups. argentophilic reaction. The background reactivity oftis-
Cassiopeiaxamachana. Thenematocystsin thetenta- sue from the oral vesicles was similar to that ofP. phy-
clesofthisscyphozoan weredivided intotwodistinct re- salis tentacle and included generalized membrane, nu-
gions. The free edges of the oral arms were white and cleus, and vesicle staining.
digitate (called "digitella" by Bigelow, 1900; "tentacles" Post-reductionalkylationofmatureandsubmaturene-
by Smith, 1937)and contained primarily ovoid nemato- matocyst capsules resulted in a virtually complete silver
cysts 12-15 Mm long, tentatively identified as euryteles. blockade,exceptintheoutermostcapsulelayeratthecni-
Theoral vesicles, ontheotherhand,containedprimarily docyte membrane interface (Fig. 1 1) and in the opercu-
small (6-9 ^m). round cnidae. Clean nematocysts ob- lum. Immature nematocysts in blockaded tissues contin-
tained from C. xamachana oral vesicles are shown in ued to exhibit a weak response to methenamine-silver.
Figure 8. The smaller cnidae contained a tubule about This response could not be distinguished from the same
1 Mm in diameter narrowing gradually (Fig. 9). Spines weak reactivity in unblocked tissues, suggesting that it
could not be measured in our SEM material, but ap- may not beduetosulfur. Theresponseofsubmaturecni-
peared uniform inTEM preparations.Thesecnidaewere dae to capsular silver was variable. In some cases, argen-
identified by Mariscal and Bigger( 1976) as holotrichous tophiliawas like that ofthe immaturecnidae; in others, a
isorhizas, and thisdiagnosis isconsistent with ourobser- greater amount ofsilver wasdeposited. In the latter case,
vations. A smaller number ofthe eurytele nematocysts silverwaseffectively blockedby post-reduction alkylation
werealsopresent in thesepreparationsand were not sep- (Fig. 1 1 inset), althoughthesilveratthecapsuleperiphery
arated by our isolation procedure. In addition, the oral couldnotbeblocked. Maturenematocystcapsulestreated
vesiclescontained developmental stages ofthese cnidae, with disulfide reducing agents alone or with iodoacetate
someofwhich were included in oursamples. orethyl maleimidealonewerestill stronglyargentophilic.
Thecapsulesofmature isorhizasfrom C. xamachana buttheywerelessintenselyblackenedand wereaccompa-
tentacular tissue were 0.4-0.5 j/m thick and were nied bygreateramountsofbackground silver.
strongly argentophilic throughout, except for the Cirrhipatheshteikcni. Clean cnidaepreparationsfrom
~0.1 Mm thick outermost region (Fig. 10). As in the whole colonies(Fig. 12) yielded spindle-shaped nemato-
small isorhizas of P. physalis, one or two electron- cysts and cylindrical spirocysts, both ofwhich were 15-
opaque annuli could be distinguished, but these were 18 MID longand about 3-4 Mm at the widest diameter. A
obscured unlessthesilverstain wasomitted. Thetubule small number of nematocysts measuring about 22 X
ofmature isorhizas was electron-opaque in part due to 5 Mm werealsopresent. Nematocystsoutnumbered spir-
nonspecific silver deposits and in part due to naturally ocystsabout 2:1 becausewholecolonieswere usedtoob-
DISULFIDES IN CNIDAE CAPSULES
a- ,0
Figures8-11. CnidaefromCassiopeia.\amacliana.
Figure 8. Cnidae isolated from oral vesiclesare primarily small isorhizas with a small percentage of
eur\teles(arrows).Scalebar= 25^m.
Figure9. SEM preparationofsmallisorhizacapsule(c)andevertedtubule(t).Spinesareappressedto
thetubuleandarenotvisible.Scalebar= 5^m.
Figure 10. Small isorhizas in various stages ofdevelopment. Immature isorhizas (ii) at top. mature
isorhizas(mi)incenter,andsubmatureisorhiza(si)atbottom. Notediffusesilverdepositsoncapsulesof
immatureisorhizasandtheelectron-opaquetubulematrix(TM)intheircenters.Centralelectron-opaque
matrix of(si) surrounds electron-lucent tubule, whereas in (mi) the tubule is electron-opaque and the
matrix(TM) iselectron-gray. Notedifferencesin capsularargentophilia: ii = diffuse,si = slightlygreater.
mi = intense.Scalebar= 1 urn.
Figure11. DTT-iodoaceticacidblockadeofmatureisorhizasblockscapsularargentophilia;cytoplasm
isstill reactive. Inset: submature capsularsilverisalsooccasionally blocked except forthe outercapsule
layer.Scalebar= 1 /im.
W. M. GOLDBERG AND G.T. TAYLOR
sn
sn
^
--".. ,-' ,
^.
^>^
17
( r -y?>>-=&..TI-2*
Figures 12-17. Cnidaefrom Cirrhipathesluetkeni.
DISULF1DES IN CNIDAE CAPSULES
tain cnide; tentacles alone would have yielded a greater with silver in one area, but in other areas only the shaft
proportion of spirocysts. Nematocysts and spirocysts wasargentophilic, orthe tubule failed tostain atall.
could not be separated and were included in the final The silver reactivity ofthe cnidae capsules in C luet-
product employed for observation and chemical analy- keni is persistent. We have tested this species (as well as
sis. Many but not all spirocysts were recovered without four other antipatharian species of the genus Anti-
capsules. Isolated nematocysts were resistant to dis- pathes see Goldberg and Taylor, 1996) using speci-
charge, and unlessthey weredisintegrated by probeson- mensthat had been fixed andstored in ethanol foryears,
ication, most were left intact by the isolation procedure. and theydisplay essentiallythe same reactivityasfreshly
Those found in the discharged state (Fig. 13) were con- collected material.
sistent with the description of microbasic b-mastigo- Post-reduction alkylationtreatmentoftissue stored in
phores(Mariscal, 1974). cacodylatebuffereliminatedall silverreactivityfrom the
The capsules of both nematocysts and spirocysts in nematocystcapsule,and most but notall reactivity from
fixed material were strongly argentophilic. Tentacular the spirocysts. Unfortunately, the background staining
cross-sections at low magnification were quite striking after blockade treatment increased considerably, partic-
when the silver-blackened outlines of the cnidae were ularly on the cell membranes. However, when we em-
comparedtotherestofthetissue(Fig. 14). Theinnerand ployed fixed material stored in ethanol rather than in
outer surfaces of the spirocyst capsule were separately cacodylate,the membranebackgroundwassubstantially
outlinedbysilverdeposits(Fig. 15 andinset). Thetubule reduced. No capsular silver was deposited in the spiro-
wall in C. luetkenispirocystswasthin, foldedat intervals cyst controls (Fig. 16). but electron-opaque deposits
intopleats,andstronglyargentophilic(Fig. 15 and inset). (Ag?) formed within the tubules. Since tubule precipi-
Thetubuleinteriorwasessentially solid,and thustypical tates were absent in all other preparations, we suspect
ofthe antipatharians examined by Goldbergand Taylor that they are artifacts in thiscase. The nematocystswere
( 1996). The unstained C. luetkenitubule contained four completely free ofCsilver deposits after blockade treat-
helically arranged bundles ofelectron-gray material. In ment (Fig. 17). In Inetkeni cnidae as in those of C.
cross-section, the bundleswere separated by electron-lu- .\amachana, treatment with disulfide reducing agents
cent, cruciform partitions, but in contrast to the tubule (DTT orthioglycolic acid) orsulfhydryl blocking agents
walland itspleats, noneofthe materialwithin thetubule (iodoacetate or ethyl maleimide) alone did not signifi-
(bundlesorpartitions)wasargentophilic(Fig. 15 inset). cantly reduce argentophilia cnidae, but increased back-
Unlike spirocysts, the nematocyst capsules were ground silverdeposits.
strongly silver-positive across the entire capsule cross-
section (Fig. 14). Nematocysttubules, ontheotherhand, Argentophiliaofimmaturecnidae
displayed an inconsistent pattern of silver deposition. The relationship between argentophilia and maturity
Often in thesame section, theentire tubule wasoutlined wasevidentin C.xamachanunematocystsbecauseofthe
Figure12. Isolatedcnidaefromwholecoloniesareamixtureofnematocystsandspirocysts.withmany
moreoftheformer.Scalebar = 100^m.
Figure 13. Discharged microbasic b-mastigophore nematocyst on tentacularsurfaceshowingshaft(s)
about the same length ofcapsule (c) with gradual transition to tubule at lower left. Tentacular cilia are
bulbouswithpointedtips,possiblyartifacts.Scalebars= 5^m.
Figure14. Methenamme-silverresponseofspirocystcapsules(sp)andmaturemicrobasicmastigophore
nematocystcapsules(mn)intentacularcross-section.Notethatcapsulesinsubmaturenematocysts(sn)are
weaklyargentophilicandthetubuleswithinaresurroundedbyanelectron-graymatrix.Maturenematocyst
tubulesarematrix-free;theircapsulesarestronglyargentophilic.Scalebar = 2^m.
Figure 15. Tangential viewofspirocyststreated asaboveshowinguniform silverreactivity ofcapsule
wall. Thetubuleand pleats(curvedarrows)arealsosilver-positive; internal portionsofthetubulearenot.
Note submature spirocyst (ss). lower left, with diffuse silver overelectron-opaque matrix; capsule wall is
indistinctandisnotargentophilic. Maturenematocyst(mn)isshownatlowerright.Scalebar=2fim.Inset:
Detail ofargentophilia in tubule wall and pleats from area ofbar-connected arrowheads in Fig. 15. Note
distinctstainingofinnerandouterlayersofthecapsule(straightarrows).Thetubulewalliscontinuouswith
thepleats(p).andbotharesilver-positive.Thetubuleinteriorisnotargentophilicandcontainsfourbundles
(b)ofelectron-graymaterialpartitionedinanelectron-lucent,cruciformpattern.Scale= 3.0xFig. 15scale
bar.
Figure 16. DTT-iodoaceticacid blockadeofmaturespirocystsblockscapsularargentophilia(arrows);
pleatsarealsoblocked;electron-opaquedepositsintubulearemostlikelyartifacts.Scalebar= I ^m.
Figure 17. Treatment as above showing complete silver blockade of mature nematocysts (mn) and
submaturenematocysts(sn).Scalebar= 1 /im.
10 W. M. GOLDBERG AND G.T. TAYLOR
9
m
-.
20
Figures 18-20. ImmaturenematocystsinPhysaliaphysalis.
Figure 18. SEM cross-section offreeze-fractured nematocyst battery showing mature cnidae at the
outer face (cleararrows). Immaturecnidaeare formed in thedeeper layers ofthe battery. Mesoglea (m)
supportstheinnerbattery:gastroderm (g)linesthecenterolthetentacle(t)andextendsintoeachbattery.
Scalebar= 25j/m.
Figure19. Toluidineblue-stainedthicksectiontakeninplaneoftrapezoid(Fig. 18)withcorresponding
locations ofmesoglea (m) and gastroderm (g). Mature cnidae at the periphery ofthe battery contain a
tubulewith no matrix. Theseoccuralongwithcapsulescontaininga basophilicmatrix(arrows), possibly
representingsubmatureisorhizas. Immaturecnidae(boxedarea)areonlyweakly basophilic. Scalebar =
25Mm.
M
Figure20. Large,immatureorsubmatureisorhizastreatedwith0.2 DTT.pH 10.3.areresistantto
depolymerization incontrasttomaturestages.Scalebar= 25
proximity of various stages of development. As noted readily distinguished from mature nematocysts by their
above,silverdeposition in thecapsulecorrespondedwith weak argentophilia. In every case, the weak staining of
the extent oftubule development. In P. physalis almost the capsules was associated with the presence of some
all of the cnidae on the outer face of the battery were remaining electron-gray matrix surrounding the tubule
mature (Fig. 18). Immature isorhizas, located deeper (Fig. 14). This matrix did not occur in mature nemato-
within the battery, were only weakly reactive toward to- cysts, suggestingthat strong argentophilia in the capsule
luidine blue and were virtually unresponsive to methe- ofthese cnidae occurs coincidentally with the complete
namine-silver reagent. However, with the development maturation ofthetubule.
of the submature stage, the tubule matrix and capsule
became strongly basophilic(Fig. 19). Because the tubule ResponseofunfixedcnidaetoDTT
matrix apparently was not rendered electron-opaque
with aldehyde fixation alone, we were unable to deter- The large isorhizas of P. physalis discharged within
mine whetherthe basophilia coincided with silverdepo- about 10-15 s ofexposure to DTT. After discharge the
sition. Thus, in the electron microscope, the submature capsule dissolved rapidly, followed by the tubule. The
cnidae could not be distinguished with certainty from time from discharge to complete solution was generally
mature isorhizas. 30-60 s. Brand el al. ( 1993)alsoobserved rapid solution
Developing spirocysts in C. luetkeni occurredjust be- in DTT, and found that P. physaliscapsulesand tubules
low the outermost, mature cysts. Submature spirocysts dissolved almost simultaneously. It should be noted,
were filled with a granular, naturally electron-opaque however,thattheseauthorsemployeddischargedcnidae
matrix surrounding (he nascent tubule. The matrix also in theirstudy. OurresultscontrastwiththoseofMariscal
containedadiffusesiI ,r-reactionproduct. Thespirocyst and Lenhoff(1969). who noted that disulfide reducing
capsule was not clear!;, developed as a double-walled agents solubilize only fully discharged nematocysts. Sol-
structure at this stage (Fig. i 5), and it exhibited affinity ubility in DTT was typical ofthe mature isorhizas only.
for silver only when the ele> \ on-opaque tubule matrix Those with clearly developed cysts but lacking a devel-
had almost completelydisappeared. oped tubule failed to dissolve in this reagent. Most of
Submature nematocyst capsules were most often lo- these immature, DTT-resistant cysts in our P. physalis
cated within clusters ofmature mastigophores and were sampleswerethe largerisorhizas(Fig. 20), butourisola-