Morphological Evidence for a Chitin-Like Glycoprotein in Penaeid Hatching Envelopes

11 Pages·1996·4 MB·English

by  P S Glas

Reference: Bin/ Bull. 191:374-384.(December, 1996)
Morphological Evidence for a Chitin-Like Glycoprotein
in Penaeid Hatching Envelopes
PATRICIA S. GLAS*, JEFFREY D. GREEN, AND JOHN W. LYNNt
DepartmentofZoologyandPhysiology, Louisiana Stale University, Baton Rouge, Louisiana 70803:
andDepartment ojAnatomy. LSUMedicalSchool, NewOrleans, Louisiana 70112
Abstract. When chitin hydrolytic enzymes (chitinase appearance of a primary extracellular coat around the
and TV-acetylglucosaminidase) were used as treatment "egg" is usually associated with activation ofthe oocyte.
during formation ofthe hatching envelope (HE) ofthe The components of this extracellular, or extra-embry-
penaeid shrimp Sicyonia ingentis. results indicated the onic, coat determine, in part, the resistance of the
presence ofcarbohydrate moieties in the envelope. Eggs embryo to invasion by pathogensand establish a micro-
exposed to theseenzymes had abnormal HEs that might environment for the developing zygote. In 5. ingentis
elevate and often collapsed. The finding that chitin syn- eggs, this extracellular coat appears about 45 min after
thase inhibitors (tunicamycin, nikkomycin Z, and poly- spawning (Pillai and Clark, 1987, 1988). The presence
oxin D)also interferedwith normal HE formation isfur- ofcarbohydrate linkages in the extracellular coat ofthe
therevidence fora carbohydrate component. The appli- crustacean egg is suggested by the abundance ofsaccha-
cation of these synthase inhibitors resulted in more rides in hatching envelopes (HE) isolated from shrimp
fragileenvelopesthatelevated andcollapsed orwereeas- (Pillai and Clark, 1990), by lectin labeling in shrimp HE
ilylostduringprocessing. Similarresultswereseen in the (Pillai and Clark. 1990), and by the localization of N-
absence ofdivalent ions (magnesium and calcium) con- acetylglucosamine in the copepod fertilization envelope
sidered critical for normal chitin formation. This mor- (Santella, 1993). Pillai and Clark (1990) demonstrated
phological evidence is indicative ofa chitin-like, linked carbohydratecomponents, in particular,TV-acetylglucos-
carbohydrate in the HE ofSicyonia ingentis. amine and mannose, in Sicyonia ingentis HE.
We
postulatethat at least threeenzymesare necessary
forassembly ofthe HE in penaeoid shrimp eggs: an oxi-
Introduction dase (Glas el a/., 1995), a transaminase (Glas et al..
1992). and a carbohydrate synthase (Glas el al.. 1993).
Oocyteactivation in the marine rock shrimpSicyonia One ofthe most abundant carbohydrate components of
ingentis takes place upon contact with seawater and is decapod crustaceans that require a carbohydrate syn-
independentoffertilization(Pillai andClark, 1987). The thaseisthepolymerof(3-1,4-/V-acetylglucosamine, more
commonly known as chitin. In this work, we show that
enzymesthat hydrolyze chitin or inhibit chitin synthase
Received2April 1996;accepted4September 1996. change HE elevation and assembly. In addition, chitin
Isl*aCndurDrre.n.tGuAdldfrBersese:zeU,.SF.LEn3v2i56r1o.nmentalProtectionAgency, 1 Sabine formation is reported tobedependent on ion concentra-
tAuthor to whom correspondence should be directed at Depart- tion and composition (Warner, 1977; Stevenson, 1985).
ment ofZoology and Physiology, Louisiana State University, Baton Clark and Lynn (1977) and Pillai and Clark (1987) re-
Rouge,LA70803-1725. ported that calcium and magnesium ions are necessary
Ahhreviations: hatching envelope. HE; ethylenediammetetraacetic forthe initial elevation ofpenaeid egg HE. In this study.
acacceieidtn,icEiasDcoiTtdhA,i;oEcGeytaTnhAayt;lee,nteraFgnIlsyTmcCio;sls-ibtoienst(efrliae-mcaetmtrihonnyoeltmrhihycolrdoaesmtchioenpre)y,Ni.TsNoE.tNMh.i;'oNc-fyllaeuntoarrtaee.s,-- wteermuseefdfecitosn-odfefdiicviaelnetntseiaownastoenr tHoEdeatsesremmibnley athnedleolnegvear-
TR1TC. tion in S. ingentis eggs. These morphological changes
374
CHITIN IN PENAEID HE 375
la
Figure 1. Eggs ofSicvonia ingenti.f 90min postspawn in artificial seawater. (a) Eggs spawned into
artificialseawaterelevatedahatchingenvelope(HE)withadistinctpervitellinespacecontainingarefractile
material. Barequals 100i*m. (b) Eggsspawned intoseawaterelevated abilayered HE with adenseouter
layerandalessdenseinnerlayer. Barequals I urn. HE.hatchingenvelope;D,denselayer;I,innerlayer;*,
perivitellinespace;R,ringvesicles;dv,densevesicles;cis,endoplasmicreticulumcisternae.
offer evidence of a chitin-like carbohydrate linkage in were removed for microscopic examination at 90 min
penaeid HE. postspawn i.e., well after normal formation ofthe HE,
a fertilization-independent event (Pillai and Clark,
Materialsand Methods 1987).
To observe the effects ofion dependency on HE as-
Gametecollectionandhandling sembly, seawaters deficient in calcium ions, magnesium
ions, or both were used. Chelator concentrations of
TherockshrimpSicyoniaingentiswascollectedbyot- 5 mM EDTA (for calcium and magnesium) or 2 mM
tertrawl from watersoffthe southern coast ofCalifornia EGTA (forcalcium)wereadded to the ion-deficient sea-
adjacent to Santa Barbara and Los Angeles. Animals water to bind extraneous calcium and magnesium ions.
weretransported incooled,aeratedtanksto Bodega Ma- Theseawaterwasadjustedwith sodium saltsto maintain
rine Laboratory, Bodega Bay, California, where they osmolarity. For ion-deficiency experiments, eggs were
were maintained with constant light in flow-through spawnedintoartificial seawater,andafter 10 minthewa-
aquaria. Spawningwasinducedbyplacingthefemalesin terwaschanged three timeswith the appropriate ion-de-
dSaprakwnneisnsgacfceomradliensgwteorethheelmdetohvoerds50ofXPi7ll0aimelmalc.ry(s1t9a8l8i)z.- pfiecnidenetntseeavweanttesr.inTehgugs,actiinvtaetrifoenrewnacesawviothideeadrl(yC,lairokn-adned-
ingdishesfilledwith artificial seawaterat room tempera-
Lynn, 1977; Lindsay etal., 1992).
ture(20C)tocollecttheeggs. Artificialseawaterwaspre-
pared according to Cavanaugh (1956), with salinity ad-
Microscopy
justed to 30%o. Controls for each experiment were
maintained in the artificial seawater at room tempera- Aliquots ofeggs from the spawning dish were placed
ture(~22C)duringtheexperiments. on clean glass slideswith coverslips supported by silicon
Inhibitorsoffungalchitinsynthesis,tunicamycin, nik- greaseto prevent desiccation and mechanical disruption
komycin Z, and polyoxin D (Calbiochem), were added of the eggs. Fresh aliquots were periodically prepared
totheeggsin seawater(pH 8.0)togive a final concentra- from the appropriate spawning dishes. Eggs were ob-
tionof1 \iM.Chitin hydrolyticenzymes,chitinase(1 ng/ served and photographed with phase microscopy during
mlor 1.5X 10~4units)(Sigma,C-1525)andTV-acetylglu- each experiment.
cosaminidase (0.05 units/ml) (Sigma, A-3189), were Samples for histology and electron microscopy were
added toeggs in seawaterat 10 min postspawn. Samples removed at 90 min postspawn and fixed with 1.2% para-
376 P. S. GLAS ET AL.
HE
2a
100pm
3a
HE
...
f
1
7
4a
CHITIN IN PENAEID HE 377
M
formaldehyde and 0.8% glutaraldehyde in 0.28 so- identify terminal sugars. Concanavalin A (Con A) for
dium cacodylate buffer in 33% seawater, pH 8.3, for 1 h mannoseand glucose, wheatgerm agglutinin(WGA) for
at room temperature. JV-acetylglucosamine and sialic acid. Lens citlinaris ag-
glutinin (LCA) for mannose, and Bandeirea simplici-
Electron microscopy folia (BS-II) for jV-acetylglucosamine (allMlectins from
Sigma Chemicals) were applied in a 0.1 phosphate
Samples fortransmission elecMtron microscopy (TEM) buffer, pH 7.2,mwiMth trace metalsadded. Control samples
were washed three times in 0.4 cacodylate bufferand contained 200 solutionsoftheappropriateblocking
post-fixed withMa final concentration of 1% osmium te- sugars (mannose, glucose, and jV-acetylglucosamine)
troxide in 0.2 cacodylate buffer for 45 min at room preincubated with the lectin for 10 min priortoapplica-
temperature. Samples were then rinsed, dehydrated tion to the section (Kiernan, 1990; Pillai and Clark,
through a graded acetone series, and embedded in a 1990). Isolated HEs were incubated in the presence of
modifiedSpurr'sresin (Spurr, 1969), substitutingQuetol the fluorescently labeled lectins for 1 h, then rinsed three
651 for DER 736 resin. Thin sections were cut with a times in phosphate buffer. Samples were observed with
diamond knife, floated on copper grids, double stained an epifluorescent microscopeequippedwith dichroic fil-
with lead citrate (Venable and Coggeshall, 1965) and terblocksforFITCand TRITC.
methanolic uranyl acetate, and viewed by TEM.
Results
Hatchingenvelopeisolationandlectin binding Spawned eggswere observed for normal HE assembly
Hatching envelopes from S ingentis eggs 90 min and elevation in the presence or absence ofchitin syn-
postspawn were isolated according to the method ofPil- thase inhibitors, chitin hydrolytic enzymes, and divalent
lai and Clark (1990) and Wikramanyake and Clark ions. Criteria for normal elevation were the continued
(1994). The eggs were hoMmogenized in amPMotter-Elveh- separation ofthe HE from the oolemma. as viewed by
jem homogenizer in 0.5 NaCl with 5 benzami- lightmicroscopy,andthepresenceofthebilayeredstruc-
dine hydrochloride and 0.1% Nonidet (Sigma Chemical ture indicative ofnormal HE assembly, as described by
Co.). Forthe homogenization, the solution wasdropped Pillai andClark (1988).
to pH 5 with a fewdrops ofHC1 to help with removal of The control eggs began elevating the HE 40-50 min
the lipid. Subsequent washes were at pH 7.8. The ho- postspawn and showed normal HE formation at 60 min
mogenate was diluted 10 times with the homogenizing postspawn (Pillai and Clark, 1987, 1988). By 90 min
solution and centrifuged at 200 Xgfor5 min. Thepellet postspawn, a retractile HE was formed around the egg
was washed repeatedly until translucent, then stored in (Fig. la). The perivitelline space contained a refractile
liquid nitrogen. material that is believed to add to the HE until the next
The frozen samples were thawed and lectin-binding extra-embryonicenvelopeisformed. ViewedwithTEM,
assays on envelopes from the same isolation procedure the HE had a thin, electron-dense outer layer and a
werecarriedoutfollowedthe methodsofKiernan ( 1990) thicker, less electron-dense inner layer (Fig. Ib) formed
and Pillai and Clark (1990). Lectins labeled with fluo- initially from components of the ring vesicles, as de-
rescein isothiocyanate (FITC) or tetramethylrhodamine scribed by Pillai and Clark (1988). The cortex ofthe egg
isothiocyanate (TRITC), both at 50 /ug/ml. were used to still contained many small, electron-dense vesicles and
Figures2-4. EggsofSicyoniaingentixtreatedwithchitinsynthaseinhibitorselevateanabnormalhatch-
ingenvelope(HE). (2a) Eggstreatedwith tunicamycinsometimesappeared normal,with an elevatedHE.
Thematerialinthepenvitellinespacelookedlinerthan inthecontroleggs. Barequals 100^m.(2b)When
viewedwithTEM,the HEaroundeggstreated with tunicamycin appearedsimilartothecontroleggs,but
thereare"holes"betweenthe HElayers(arrowhead). Themicrograph isintwosectionstoallow agreater
viewofthecortex oftheegg. Barequals I pm. (3a) Eggstreatedwith polyoxin Delevated envelopesthat
appearedtohaveanormal HE.(3b)When viewedwithTEM,eggsin polyoxin Dhadabnormal HEswith
clearspacesor"holes" intheinnerlayer(arrow)andbetween thetwoenvelope layers(arrowhead). Large
yolkgranules(y)displacedtheringvesiclesandmitochondriatothecortexoftheegg.(4a)Eggstreatedwith
nikkomycinZelevatedHEs(arrowhead)thatremainedclosetotheegg.(4b)Eggsin nikkomycinZviewed
with TEM appearedtohave lost the HEsin the fixationandembeddingprocess. Thecisternaeanddense
vesiclesweresituatednormally,buttheringvesicles(R)werenotaspackedasinthecontrols.HE,hatching
envelope; D.denselayer; I, innerlayer;*,perivitellinespace; R, ringvesicles;dv,densevesicles;cis,endo-
plasmicreticulumcisternae;y,yolkplatelets.
378 P. S. GLAS ET .11.
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CHIT1N IN PENAEID HE 379
large ring vesicles tightly packed with ring-shaped ele- exocytosedringvesiclesintothe HEwasincomplete,and
ments. These vesiclesappearedsimilartothoseobserved the ringelements remained conspicuous in the perivitel-
by Pillai and Clark (1988) during the early formation of line space. The ER cisternae and the mitochondria were
the HE. Small cisternae ofendoplasmic reticulum (ER) less conspicuous in the peripheral cortex (Fig. 2b) com-
occupied much ofthe cortical region (Fig. Ib). Vesicles pared to control eggs. In TEM preparations of eggs
loosely filled with ring elements and mitochondria in treated with polyoxin D, the HE was abnormal and col-
close association with Golgi complexes were frequently lapsed to the oolemma surface (Fig. 3b). The flocculent
observed. For a more detailed description of early HE inner layer was interrupted by many large electron-
formation, referto Pillai andClark (1988, 1990). translucent areas(holes). Theouterlayerand innerlayer
hadaseriesoffineelectron-translucentspacesseparating
Chitin synthesisinhibitorsblocknormalHEformation the layers. The cortex ofeggs treated in polyoxin D, un-
like that ofcontrol eggs, was filled with prominent yolk
Eggswere treated with three chitin synthase inhibitors, platelets. The peripheral relocation ofthe yolk appeared
tunicamycin, nikkomycin Z, and polyoxin D. In the to compressthe ER and mitochondria in the eggcortex.
treated eggs, HE formation, elevation, or both were ab- Small electron-densTeEvMesicles were not seen in the pe-
normal. Eggs treated with tunicamycin elevated HE in ripheral cortex. In micrographs, eggs treated with
twooutofthreetrialswhenassayedwith light microscopy nikkomycin Z lost the HE during processing (Fig. 4b),
(Fig. 2a). In some eggs, the HE remained close to the egg indicating an extremely fragile HE. Numerous vesicular
surface so that the perivitelline space was obscured (not structures were adjacent to the egg surface. The cortical
shown). Granular material finer than that observed in region resembled that ofan egg during early HE forma-
control eggs was seen in the perivitelline space. Eggs tion and elevation, with many small electron-dense vesi-
treatedwith polyoxin Dhadenvelopesthatappearedsim- clesand large vesiclescontaininga few ringelements.
ilar in structure to thecontrol eggwhen viewed with light
microscopy (Fig. 3a). In eggs treated with nikkomycin Z, Chitin hydrolyticenzymesinhibit normalenvelope
the HE remained close to the egg surface (Fig. 4a) and assembly
theperivitellinespacewasfilledwithafine material, often
makingit difficultto visualizetheseparation from theegg Sicyonia ingentis eggs were treated with the chitin hy-
surfacewhen viewed with lightmicroscopy. drolytic enzymes (chitinase and /V-acetylglucosamini-
Eggs treated with tunicamycin and examined with dase) at 10 min postspawn and observed until 90 min
TEM hadabilayerHEasin control samples, butthetwo postspawn. Chitinase-treated eggs formed HEs that ini-
layers had electron-translucent areas, or "holes," be- tially elevated farther from the eggs than in the controls,
tween the thin electron-dense outer layer and the floc- then collapsed (Fig. 5a). Hatchingenvelopeswere not ap-
culent inner layer (Fig. 2b). This condition resembled parent in yV-acetylglucosaminidase-treated eggs in three
normal eggs during early elevation (i.e.. 60 min out ofthreetrials(Fig. 6a). When bothTV-acetylglucosam-
postspawn), suggestinga delay in HE formation (see Pil- inidase and chitinase were added to the spawning media,
lai and Clark, 1988, for comparison). Incorporation of again no HEs were discernible around the eggs (Fig. 7a),
Figures5-7. EggsofSicynniaingentixtreatedwithchitinolyticenzymesdonotelevateanormalhatch-
ingenvelope(HE). (5a)Eggstreated withchitinaseelevated HEs,buttheenvelopesappearedthinnerthan
thecontrol envelopesand usually collapsed to the oolemma surface. Barequals 100^m. (5b) The HE of
eggstreatedwithchitinaseappearedtohaveformedwiththetwolayers,buttheinnerlayerwasnotasthick
asin control eggs. Thedense layer(D) wasseparated from the innerlayer(I)by small holes(arrowhead).
Thepenvitelline space(*)contained liner material than seen in thecontrol eggs. Endoplasmic reticulum
cisternaeanddensevesiclestilledtheeggcortex,butnopackedringvesicleswereseen nearthecortex. Bar
equals 1 /im. (6a) Eggstreated with A'-acetylglucosaminidasedid not haveelevated HEs.(6b)A'-acetylglu-
cosamimdase-treatedeggsdidnotform normal HEs.Densevesicleswereseeninthecortex,andthepacked
ringvesicleswerepresent, buttheintegrityol thecortical regionwasdisrupted. Barequals 1 ^m.(7a)Eggs
treated with both chitinaseand A'-acetylglucosaminidasedid notelevate normal HEs. Theeggsappeared
swollenwithahighly refractivecortex.(7b) Eggstreated with both chitinaseand A'-acetylglucosaminidase
did not haveHEswhen viewedwithTEM.Theexterioroftheoolemmaresembledthesurfaceofaneggin
early HE formation. The large ring vesicles had only a few ringelements in them. Again, the structural
integrityofthecortexappeareddisrupted. Barequals 1 ^m. HE.hatchingenvelope:D,denselayer,I,inner
layer;*,perivitellinespace;R.ringvesicles;dv,densevesicles;cis,endoplasmicreticulumcisternae;y,yolk
platelets.
380 P. S. GLAS ET AL.
HE
8a
100|jm
HE
dv
D
9a 9b
i
*-.,
HE,
1pm
10a lOb
CH1TIN IN PENAEID HE 381
and the egg appearance resembled that of a newly (Fig. 9a), although the HE did not appear to elevate as
spawned egg (see Filial el al., 1988). These eggs often ap- far from the egg as in the controls. Granular material
pearedswollen, witha highly refractivecortex. Eggs in N- similartothat in control eggswasvisible in the perivitel-
acetylglucosaminidasealone orboth chitinase and A'-ace- linespace. Noelevated HEwereobserved in eggstreated
tylglucosaminidase were very fragile and broke easily with seawaterdeficient in both calcium and magnesium,
when pipetted orswirled vigorously in thespawningdish. although a thin HE precursorcould be seen coveringthe
With TEM, the HEsofthe chitinase-treated eggswere second polar body (Fig. lOa). The eggswere very fragile,
bilayered, similar in appearance to control envelopes breakingeasily with handling.
(Fig. 5b). The ER cisternae were as numerous as in con- Examination ofeggs from calcium-deficient seawater
trol eggs, but appeared more electron-translucent, sug- withelectron microscopyshowedan HEwiththe bilayer
gesting lack of material content. Small dense vesicles morphology, but the envelopes had collapsed to the
were abundant, but the large packed ring vesicles were oolemma (Fig. 8b), again suggesting lack oftanning of
not seen. TEM ofjV-acetylglucosaminidase-treated eggs the envelope. Small dense vesicleswere abundant in the
showed athin, unorganized layerofmaterial resembling cortex of the egg, but the mitochondria and cisternae
the HE precursor (see Pillai and Clark, 1988) on the ex- were misshapen by yolk platelets. Eggs in magnesium-
terior of the egg membrane (Fig. 6b). The eggs lacked deficientseawatershowed HEswithathinbilayeredcon-
structural integrity, suggesting eventual cell death. Vesi- struction (Fig. 9b). However, the HE had large electron-
cleswith ringstructureswere smallerthan in the control translucent areas ("holes") between the two layers and
eggs. The small dense vesicles were similar to those in within the innerlayer. The ringelementsdid not appear
control eggs. When viewed with electron microscopy, to be incorporated into the HE. The ER cisternae were
eggs treated with a combination ofboth enzymes had a prominent in the cortex. Ninety min postspawn eggs
thin, unorganized area visible external to the oolemma treatedwithcalcium-and magnesium-deficientseawater
(Fig. 7b); this area resembled the HE precursor. Dense showed HEs that were morphologically similar to HEs
vesicles were abundant in the cortex, and the large vesi- during early formation (Fig. lOb) (see Pillai and Clark,
clescontained onlya few ringelements. Theleached ap- 1988, for comparison). The ring vesicles were not as
pearanceagainsuggestedextremedisruptionofcell func- packed as in the controls, and the cortex appeared sim-
tion leadingtocell death. ilar to that ofa normal egg during the period immedi-
atelybefore normal HE elevation and assembly.
Iondeficient seawatersaffectnormalenvelopeassembly
Shrimp-isolatedHEslabel with $-glucosidespecific
Inthefollowingexperiments,eggswerespawned inar- ledins
tificial seawater, washed three times in the appropriate
ion-deficientseawaterat 10 min postspawn, andallowed Envelopes isolated from eggs at 90 min postspawn
todevelop. In calcium-deficient seawater, the HE, asob- were incubated with fluorescently labeled lectins to de-
served with phase microscopy, elevated farther from the tectthepresenceofcarbohydrates: ConcanavalinA(Con
eggsurfacethan incontrols(Fig. 8a). Theeggwasusually A) for mannose and A'-acetylglucosamine, wheat germ
eccentric within the HE, and the HE would collapse agglutinin (WGA) for A-acetylglucosamine, Lens culi-
wheneggswerehandled. ElevationofHEbyeggsin mag- narisagglutinin (LCA)formannose,andBandeireasim-
nesium-deficient seawater appeared relatively normal plicifolia(BS-II)forA'-acetylglucosamine. Untreatediso-
Figures8-10. Eggsincubatedinion-deficientseawaterdonotelevateanormalhatchingenvelope(HE).
(8a)Eggstreatedwithcalcium-deficient seawaterhad an HE thatappearedtoelevate fartherfrom theegg
thaninthecontrols. Barequals 100^m.(8b)Eggstreatedincalcium-deficientseawaterhadan HEthatwas
collapsedtotheoolemma,althoughabilayeredstructurecouldbeseen. Yolkandlipiddropletswereclose
totheoolernma. Barequals 1 ^m.(9a)Inmagnesium-deficientseawater,theHEdidnotelevateasfarfrom
theeggasinthecontrols.(9b)TheHEinmagnesium-deficientseawaterwasfilledwithelectron-translucent
areas,or"holes,"betweenthetwolayersoftheHE(arrowhead).Theinnerlayer(I)wasnotaswellorganized
asinthecontrolorincalcium-freeseawater.Themicrographisintwosectionstoallowagreaterviewofthe
cortex oftheegg. Barequals 1 Mm. (lOa) In calcium- and magnesium-deficient seawater. the HE did not
elevate from theeggsurface although a thin HE precursorcould be seen coveringthe second polarbody
(pb).(lOb)Incalcium-andmagnesium-deficientseawater,anormal HEwasnotformed.Ontheexteriorof
theoolemmawasathinlayer,probablytheHEprecursor(HE,),butthedenselayerandinnerlayerhadnot
formed. Barequals 1 ^m. HE,hatchingenvelope;D,denselayer;I,innerlayer;*,perivitellinespace;R,ring
vesicles;dv,densevesicles;cis,endoplasmicreticulumcisternae;y,yolkplatelets.
382 P. S. GLAS ET AL.
lates showed no fluorescence (not shown). LCA and
ConAstronglylabeledtheisolated HEs(Figs. 11 and 12).
WGA (Fig. 13)and BS-II (Fig. 14) showed much weaker
staining, and BS-II showedonlyaslightreaction withthe
isolated envelopes.
Discussion
The focus ofthis study is on the formation and eleva-
tionoftheshrimp HEafterexposureoftheeggstochitin
hydrolytic enzymes or chitin synthesis inhibitors. How-
ever, the potential role ofthe cortex in the synthesis of
any chitin-type carbohydrate cannot be ignored. Thus,
the morphology ofthe cortex is an important aspect of
HEformation. Thecortical regionofthepenaeidshrimp
eggat 90 min postspawn hastheclassic featuresofabio-
logicallyactivecell abundant mitochondria, endoplas-
mic reticulum, and cisternae. At least two types ofvesi-
cles with morphologically different characteristics are
present, suggesting additional synthetic activity. Obser-
vations in this laboratory have shown that the HE con-
tinues to have material added to it until the next enve-
lope is constructed at 90-1 10 min postspawn. Conse- Figures 11-14. Fluorescent micrographsshow labelingofisolated
quently, biochemical changes such as cross-linking hatching envelopes (HEs) from Sicyonia ingenlis eggs at 90min
(tanning or hardening) ofthe HE and incorporation of WpoGstAspawn. Labelingasfollows:(II)LCAlectin;(12)ConAlectin;(13)
newmaterial intothe HE maycontinue until much later HEstrleeactteidn;w(i1t4h)WBSG-IAI leacntdin.BSN-oItI.eUtnhetrreealatteidvecdoentcrroelasHeEisnlsahboelwiendgfnoor
than originally expected and ultimately result in the ap- backgroundfluorescence. Barequals50^m.
pearanceofchitin-likecomponents in the HE. Thethree
experimental approaches used in this study, i.e.. inhibi-
tory, hydrolytic, and ionic treatments, resulted in visible
changes in the morphology and assembly ofthe HE, as Nikkomycin Z is a competitive inhibitor ofchitin syn-
well as in the morphology of the cortex. These results thase in fungal cell walls (Cabib, 1987). Although these
weredramaticallydifferent from the normal sequence of antibiotics are reported to inhibit chitin synthetic path-
events described in earlier investigations (Pillai and ways, the possibility ofaction on other related or unre-
Clark, 1988, 1990; Lynn el ai. 1991, for review). Inter- lated metabolic pathways in decapod development can-
estingly, the treatments often result in eggs that appear not bedismissed.
to havebeen arrested in earlierstagesofdevelopment. Treatment ofthe eggs with two enzymes that are con-
When eggsare treated with chitin synthase inhibitors, sidered to be part of the chitinolytic pathway results,
HEelevation occurs, butthestructureofthe HE and the again, in abnormal elevation and assembly ofthe HE.
morphology ofthe cortical region ofthe egg are affected With chitinase treatment an HE iselevated and appears
bythesynthaseinhibition. Thecollapseorlossofthe HE somewhat normal in structure, but the collapse of the
during handlingsuggestsextreme fragility orsusceptibil- HE suggests abnormal hardening (i.e.. cross-linking) of
ity to chemical stress. The severity ofthe morphological the envelope. Although preliminary assays indicated
abnormalities differs with the actions ofthe chitin syn- some proteolytic activity in the chitinase enzyme, pro-
thaseinhibitors. Thisisnotsurprisingbecausethe inhib- teolytic inhibitors could not be simultaneously used be-
itors are known to react differently between the insects cause of their deleterious effect on HE elevation and
andothercrustaceans(Cabib, 1987, 1991;Cohen, 1987). morphology. In addition, the chitinase results indicate
Tunicamycin is an antibiotic that blocks the formation relatively subtle changes in HE formation in contrast to
ofprotein-carbohydratelinkagesoftheA'-glycosidictype what occurs in proteolytic treatments ofthe HE. Thus
(Duskin and Mahoney, 1979). Polyoxin D is a specific while proteolytic effects cannot be ruled out, the results
inhibitoroffungalchitinsynthasethat isknowntoresult ofthe chitinase treatment are not consistent with such
in malformed endocuticular layers in the larvae of the effects. Eggs treated with TV-acetylglucosaminidase, the
butterfly, Pierisbrassicae, acabbagepest(Cohen, 1987). chitinolyticenzyme, appeared nottoelevatean HE. The
CHITIN IN PENAEID HE 383
chitinolytic enzymes do not appear to degrade the HE function ofthiscell. Furtherexploration ofthe enzymes
from the outside, as might be expected; instead, they and resulting events during this period could offer new
seem to affect the assemblyperse. Since the pH optima insightsintothemechanicsofembryoprotectionandac-
for chitinase and yV-acetylglucosaminidase are 6 and 4, tivityduringearlydevelopment.
respectively(Muzzarelli, 1977), hydrolyticactivity is un-
likelytooccuratthepHofseawater(pH 8.0).Thus,these
Acknowledgments
enzymes are probably binding to substrate and
effectively blockingfurtherpolymerization. The leached The authors express their appreciation to Dr. Wallis
appearance ofthe cortex ofeggs treated with TV-acetyl- Clark and his students and colleagues for lab space at
glucosaminidase suggests that one mode ofaction may Bodega Marine Lab (UC-Davis) and for their patience
be the disruption ofthe cortical architecture. Whether andassistancein theprocurementofS. ingentis. Wealso
this disruption interferes directly with the release of a thank Eleanor Uhlinger for her assistance in obtaining
necessary substrate or enzyme or indirectly through cell reference materialsand Ron Bouchard and Cindy Henk
HE
death isunclear. However, theassembly ofthe in the ofLSU Life Sciences Microscopy Facility for assistance
presenceofchitinasealonesuggeststhattheW-acetylglu- with the microscopy and photography. Rachel Arnold,
cosaminidase is necessary forthe lack ofHE formation, Kathryn Kreimborg, and Shayd Coots were invaluable
much as the combination of enzymes is necessary for in theirassistancewith the laboratory work.
completedegradation ofchitin. This work was supported in part by Louisiana Sea
Changesin ioniccompositionoftheseawaterafterini- Grant #NA89-AA-D-SG226 project #R/SA-1 to JWL
tialegg-sperm interaction hasoccurred alsoaffecttheas- and JDG and Sigma Xi Grant-in-Aid of Research to
sembly ofthe HE. Deficiency ofMg+2 and Ca+2 ions re- PSG.
sultsin eggswith abnormal HE formation, cortical com-
position, or both (Clark and Lynn, 1977; Pillai and
Clark, 1987). In the eggs treated with ion-deficient sea- LiteratureCited
water, the holes between the two HE layers and within
Cabib,E. 1987. Thesynthesisanddegradationofchitin. Pp. 59-101
the innerlayersuggestlackofincorporation ofmaterials in Advance'sin EniymologyamiRelatedAreasofMolecularBiol-
into the HE. In particular, the packed ring vesicles seem ogy, Vol. 59.Wiley,NewYork.
to be affected by these ionic deficiencies, as treated eggs Cabib, E. 1991. Differential inhibition ofchitin synthetase I and 2
were lacking the large number of packed ring vesicles from Saccharomyces serevisiae by polyoxin D and nikkomycins.
found in the control eggs. The morphology ofHioEn-de- CavAanntaiumgihc,roGb..MA.ge(netds.)Ch1e95m6o.lherF.or3m5u:l1a7e0-a1n7d3.Ale/hodsoftheMarine
pleted eggs suggests an interruption or delay in for- BiologicalLaboratoryChemicalRoom, 6thed.. Marine Biological
mation. Assuggested forearly egg activation (Clark and Laboratory.WoodsHole.MA.
Lynn, 1977; Pillai and Clark, 1987), Mg2+ appearsto be Clark,W.H.,Jr.,andJ.W.Lynn. 1977. A Mg*+dependentcortical
more important than Ca2+ for normal envelope assem- reactionintheeggsofpenaeidshrimp./ Exp. Zool. 200: 177-183.
bly. Thepresenceofatleastoneoftheseionsisnecessary CoheRne,v.EE.nt19o8m7o.l.3C2h:i7tlin-9b3i.ochemistry: synthesisand inhibition.Ann.
fornormal HE assembly. Duskin,D.,andVV.C.Mahoney.1979. Biologicalactivitiesofthetwo
Thelectin study showed somedifferences from results majorcomponentsoftunicamycin./Biol.Chem 254:6572-6576.
reported by Pillai and Clark(1990). However, thediffer- Glas,P.S.,J.D.Green,andJ.W.Lynn.1992. Enzymeinvolvement
ence in the time points ofthe samples may account for in elevation and tanning ofthe hatching envelope ofa penaeid
sthoomueghofgltuhceosdeisacnrdepamnacnineso.seThreesirdeusuelstasresupgrgeessetntthianttahle- Glassf,hrroP.immSpp.e,onJva.euiDmd..sGPhrrreoice.mn,pLaea.xnhdAicbJai.td.Wc.hSictiLi.ynn-5l5ni:.ke4129par9.o3p.ertiHeast.cMhoiln.gBeinovle.lCoeplel.s
HE, labeling for jV-acetylglucosamine oligomers by BS- 4:378a.
WGA
II and suggested either less accessibility or less Glas,P.S.,J.D.Green,andJ.W.Lynn. 1995. Oxidaseactivityasso-
abundance compared to the results of Pillai and Clark ciatedwiththeelevationofthepenaeoidshrimphatchingenvelope.
Biol. Bull. 189: 13-21.
(19T9h0)e.results ofthis study, together with previous dem- Kierannadn,PrJa.cAti.ce199P0e.rmalglo^tnolPorxesisc,ulOx&foHrids.tochemical.Methods: Theory
onstration ofan oxidase (Glas el ai, 1995), offer argu- Lindsay,L.L.,P.L.Hertzler,andW.H.Clark,Jr.1992. Extracellu-
mentforthepresenceofachitin-like,orsimilarlylinked, lar Mg:+ inducesan intra-cellularCa:+ waveduringoocyteactiva-
carbohydrate component in the HE ofpenaeid shrimp. tion in the marine shrimp. Sicyoma nixcntis De\- Biol 152: 94-
The morphology ofthe penaeid egg during early devel- 102.
Lynn,J.W.,M.C.Pillai,P.S.GIas,andJ. D.Green.1991. Compara-
otipvmeecneltl.hTahseallcotnhsetrchuacrtaicotneroifsttihcesHofEaabnidocthheemiecxatlrlay-eamc-- atievoeidmeoar.pPhpo.l4o7g-y6a4nidnpFhryosnitoileorsgyinofShegrgimapctRievsateiaornchi.nMse.leAc.teDdavpiedn--
bryonic envelopes that are formed later is a dynamic sonandW.J. Dougherty,eds. Elsevier,Amsterdam.