Experimental and Histological Studies of Four Life-History Stages of the Eastern Oyster, Crassostrea virginica, Exposed to a Cultured Strain of the Dinoflagellate Prorocentrum minimum

16 Pages·1995·6.3 MB·English

by  G H Wikfors

Reference: Biol Bull 188: 313-328. (June, 1995)
Experimental and Histological Studies of Four Life-
History Stages of the Eastern Oyster, Cmssostrea
virginica, Exposed to a Cultured Strain of the
Prorocentrum minimum
Dinoflagellate
GARY H. WIKFORS1 AND ROXANNA M. SMOLOWITZ2
^National Oceanic andAtmosphericAdministration, NationalMarine Fisheries Service, Northeast
Fisheries Science Center, Milford, Connecticut 06460; and2LaboratoryforMarineAnimal Health,
University ofPennsylvania, MarineBiological Laboratory, Woods Hole. Massachusetts 02543
Abstract. Effects of the dinoflagellate Prorocentrum Introduction
minimum (strain EXUV) upon four life-history stages of
the eastern oyster embryos, feeding larvae, newly set Harmful algalbloomsareviewedasan increasingthreat
spat, and juveniles were investigated in laboratory ex- to utilization of living marine resources (Hallegraeff,
posure studies. Embryonic development was notaffected 1993). Studiesofinteractionsbetweendinoflagellatesand
significantly by living, heat-killed, or sonicated cells, or molluscan shellfish have focused mainly on the accu-
by growth-medium extracts from P. minimum cultures. mulation ofmammalian neurotoxinsbymolluscsfeeding
Feeding larvae, however, showed poor growth and poor on toxic dinoflagellates(Sakamoto el ai, 1987). More re-
development of the digestive system when fed P. mini- cently, the detrimental effects oftoxic dinoflagellates, as
mum, as compared with larvae fed Isochrysis sp. (strain well as the effects ofsome algae that produce no known
T-ISO). Growth oflarvae fed mixed P. minimum + Iso- mammalian toxins, upon the shellfish themselves have
chrysisdietswas intermediate. Larvae and newly set spat been identified(Shumway, 1990). Responsesrangingfrom
that had been fed adiet of'/j P. minimum + %Isochrvsis reduced filtering to increased metabolic rates, paralysis,
exhibiteddistinctivechangesin digestive-system anatomy. and mortality havebeen noted for molluscs feeding upon
Spat showed an abnormal accumulation of lipid in the natural populations ofdinoflagellates or cultured strains
stomach epithelium. Absorptive cells in the digestive (Gainey and Shumway, 1988). It has, in fact, been sug-
glands of both larvae and spat contained accumulation gestedthatdetrimental effectsupon shellfish byanumber
bodies, often with a laminated, fibrous appearance in ofphytoplankton taxa maybeundetected ratherthan un-
common
preparationsfortransmissionelectron microscopy. These (Parry el ai, 1989).
accumulation bodies were PAS (periodic acid-Schiff) Among dinoflagellate taxa implicated in toxic effects
positive and may correspond to autolysosomal bodies are members of the genus Prorocentrum. Two benthic
within P. minimum cells. Juvenile oysters developed the Prorocentrumspecies, P. limaandP. mexicanum, produce
ability to digest P. minimum, but only after a refractory ciguatoxin, which accumulates in tropical finfish food
period ofabout 2 weeks, duringwhich mostP. minimum chains and renders apex predators unfit for human con-
was filtered but rejected as pseudofeces. The linking of sumption (Steidinger, 1983). Therearehistorical accounts
accumulation bodies within absorptive cells of oyster of P. minimum and its taxonomic equivalents P.
digestive diverticula with dinoflagellate autolysosomal mariae-lebouriae, Exuviaella mariae-Lebouriae, and P.
bodies suggests a mechanism by which some dinoflagel- triangulatum(Dodge, 1982) causingtoxiceffectsin both
lates interfere with feeding in phytoplankton grazers. shellfishandhuman consumersofshellfish harvested from
P. minimum bloom water (Shumway, 1990). One strain
ofP. minimum that is cultured in a number of labora-
Received 1 September 1994;accepted 21 February 1995. tories, cloneEXUV (CCMP1329), yielded negative results
313
314 G H WIKFORS AND R M. SMOLOWITZ
in previous(Schmidtand Loeblich, 1979)mousebioassays gimes were specified according to packed-cell volume
forwater-soluble mammalian toxins (PSP) and in assays (specified for larvae or postset below). Cell counts were
carried outduring the present study(S. E. Shumway, pers. made in an Improved Neubauer hemocytometer.
comm.). Despite the negative finding for mammalian
toxin in this strain, we demonstrated previously that Oyster rearing
EXUV
cultured in our laboratory did not support the
Broodstock oysters from the Milford Laboratory were
growth ofjuvenile northern quahogs, Mercenaria mer-
spawned by warm-water stimulation (Loosanoff and
cenaria, and was acutely toxic to juvenile bay scallops, Davis, 1963); 5 females and 8 males contributed gametes
Argopecten irradians (Wikfors and Smolowitz, 1993).
EXUV to the cohort used in experiments. All oyster rearing was
Complete mortality of scallops exposed to the
strain occurred within 1-4 weeks of first exposure. Af- in Milford Harbor seawater (27-28%o salinity) that was
temperature-adjusted to 25 C, filtered to 0.5 ^m, and
fected scallops had severe attenuation ofepithelial cells
passed through a flow-through ultraviolet sterilizer and
associated with absorptive-cell necrosis and sloughing of
cells into central lumens. Large melanized hemocyte clots an activated-carbon organic-removal cartridge. For the
experiment with embryos, fertilized eggs weredistributed
were present throughout the open vascular system; these
into 1-1 polypropylene beakers at a concentration of
histologic observations are strong evidence for the pres-
ence ofa molluscan enterotoxin in EXUV. 20 ml"1. Embryos were exposed to experimental treat-
Because P. minimum is a normal component of the ments (see below) within 2 h offertilization. Remaining
embryos were incubated in seawater treated as above for
summertime flora in thecoastal watersofthe northeastern
United States (Marshall, 1980; Sellner et ai, 1993; Wik- 48 h before experiments were begun with feeding larvae.
Subsamples of 48-h larvae were collected on a 36-^m-
fors, inprep.), itseemed possiblethat nativeoysters, which mesh monofilament nylon screen and distributed into 1-
spawn during summer, could be exposed to this dinofla-
gapelnladlyagtseeooagmtreaapnrhyoilcseteaixgnteetnohtfeotfhreePicrrroulriioftecmeheninstttrousrmyu.cpcoTepisusmliaontfgi,oonydssetncesorisutlyid,f a1innbdefaekePedarivsnlgoatveaaxpcleourntihcmeeerninttr(saMtiwileofrnoerodfre2sa0trremadiPn'o.nMLOaaNrdivOeat)eonftohtTr-ouIusSgeOhd
metamorphosisand then grownon natural phytoplankton
therearedetrimentaleffectsatparticularlife-historystages. amended with cultured algae (mostly Tetraselmis
Therefore, the present study was undertaken to expose TTM
maculata strain and Pavlovagyransstrain 93, with
ofyesetdeirnsg altarvfaoeu,rnleiwfle-yhissettorsypats,taagneds jupvreenfileeesdingtoecmublrtyuorse,d small amountsofdiatomsand otherflagellates). Fourdays
P. minimum, both alone and in combination with a before use injuvenile feedingstudies, oysterswere placed
known "good-food" alga, Isochrysis sp. (strain T-ISO). in filtered seawater to empty the digestive systems.
Survival, development, growth, and histologic condition
Experimental design
wereevaluatedandcompared with thoseofstarvedoysters
andoystersfedT-ISOalone an algaldiet knownto sup- Oyster embryos were exposed to a variety oflive and
port normal growth and development (Wikfors, unpub.). killedcellsandextractsfromP. minimumand thecontrol
alga, T-ISO, shortly after fertilization ofeggs. Live algal
Materials and Methods cultures were added, along with the culture medium in
which they weregrown, asaliquotsprovidingthe number
Phytoplankton culture
ofcells given as a first feeding according to the regime of
The EXUV strain of P. minimum (CCMP1329) was Rhodesand Landers(1973); i.e., 0.005 ml packed cellsT'
obtained from the Provasoli-Guillard Center forthe Cul- larval culture containing 20 larvae ml"1 for T-ISO, this
;
ture of Marine Phytoplankton; Isochrysis sp. strain T- was60 X 1 6 cells P1, and for EXUV, 3.9 X 106 cells T1.
ISO was in the Milford Microalgal Culture Collection. Equal amounts ofboth algal strainswere subjected tothe
Both axenicalgal strainswerecultured in enriched natural following treatments before being added to embryo sus-
seawater medium, E formulation (Ukeles, 1973), in 16-1 pensions: (1) removal ofcellsWby filtration to 0.22 /urn, (2)
carboy assemblies housed in a temperature-controlled sonication for 5 min at 250 with 50/50 timed pulse,
(20C) room with constant illumination of 500- or(3) heatingto 80C for 30 min. Inaddition totheabove
600 nE m~: s~' from cool-white fluorescent lamps. Cul- treatments, live and sonicated EXUV culture, and me-
tures were managed semi-continuously, with daily har- dium from EXUV culture, wereaddedat 5 timesstandard
vestsofsufficientvolumestosatisfyexperimental regimes volume. Seawater-only and sterile algal medium controls
and weekly replacement ofharvested volumes with ster- were included as well. All treatments were in triplicate.
ilized medium. Culture densities were determined as Embryos were incubated on a temperature-controlled
packed-cell volumes by centrifugation in specially mod- water table (25C) for 48 h, and then evaluated micro-
ified Hopkins tubes (Ukeles, 1973), and daily feeding re- scopically for effects upon survival and development.
OYSTERS EXPOSED TO A DINOFLAGELLATE 315
Counts oflive-normal, live-abnormal, dead-normal, and anda full ration ofEXUV was9.27 X 106cellsperoyster;
dead-abnormal larvae were made for all treatments. cell numbers in percentage diets can be calculated from
Larval oysters, which had been permitted to develop these values. Seawater flow through the chambers was
for 48 h in filtered seawater, were distributed into 1-1 continuous, except forthe4 h afterfeeding, duringwhich
beakers for larval feeding experiments, also incubated on flowwasstopped. Fluorometermeasurementsshowedthat
the 25 C water table. Larval populations in triplicate all algal food suspensions werecleared within 2 h offeed-
beakers were given the following daily feeding regimes: ing. Each week, for 6 weeks, oysters were removed from
(1) EXUV, (2) 'A EXUV + % T-ISO, (3) % EXUV + 'A chambers and cleaned with a pressurized spray of sea-
T-ISO, (4) T-ISO, and (5) unfed. Daily rations were ad- water; with theaid ofimage-analysis softwareonan IBM-
justed according to packed-cell volume, following the compatible microcomputer, shell lengths were measured
progressively increasing schedule ofRhodes and Landers from video images.
(1973). Concentrations (cells per liter) of T-ISO and
EXUV alone on the first day offeeding are given in the
Histology
previousparagraph; cellsin mixed dietsand in increasing
rations can be calculated from these. Three days each Subsamples of48-h larvae, feeding larvae, and newly
week, larvae were collected on a 15-nm monofilament set spat were processed in plastic (Embed 812, Electron
nylon screen, washed with a pressurized flowofseawater, Microscopic Sciences). Briefly, samples fixed in 1% glu-
and resuspended in newly filtered seawater. At this time, taraldehyde/4% formalin in 28%o NaCl (1G4F) (Howard
subsamplesoflarvaewerecountedonaSedgewick-Rafter and Smith, 1983) were rinsed with phosphate buffer, sec-
slide under a dissecting microscope and measured (shell ondarily fixed in 1% osmium, and hydratedtounbuffered
length) on a compound video-microscope using image- water. Oystersweredecalcified in 2%ascorbicacid in0.9%
analysis PC-computersoftware. In vivofluorescence, pH, sodium chloride at pH 2.3 for 15-20h, followed by a
and salinity ofspent larval-culture water were measured rinse ofunbuffered water. Sampleswereconcentrated be-
every 3 days as well; water for these samples was poured foreremoval ofeach solution bycentrifugingat 1300 Xg
gently through a 36-mesh nylon screen to minimize the for3 min, followedbyremoval ofthesupernatant. Oysters
effect of fecal material upon the fluorescence measure- then were embedded in 3% agarose (aqueous). Enbloc
ments. Subsamples oflarvae were taken periodically and staining was done in 2% uranyl acetate (aqueous) over-
fixed for histological examination; results for samples night. Staining was followed by infiltration, embedment
taken 8 days after spawning are reported. Larvae fed T- in Embed 812, and polymerization by standard methods.
ISO, EXUV, and the 'A EXUV + 2A T-ISO diet were ob- Thick sections cut from the plastic-embedded samples
served live, 60 min after feeding on day 14, underepiflu- werestainedwith 1% methylene bluein 1% borax(sodium
orescence microscopy (Babinchak and Ukeles, 1979) to borate) and 1% azure II. Ultrathin sections were stained
determine iflarvae were ingestingand digestingalgal cells. with 2% uranyl acetateandcounter-stainedwith Reynolds
In cultures that produced pedi-veligers, whole oyster lead-citrate. Thick sections were examined and photo-
shells were provided as setting substrate, and setting suc- graphed usingan Olympus BH-2 photomicroscope. Thin
cess was determined by counting spat on shell surfaces. sections were examined on a Zeiss 10 transmission elec-
Newly set spat continued to receive the diet provided to tron microscope.
them as larvae. Spat were sampled for histological ex- Juvenile oysterswere fixed //;situby placing the oyster
amination 5 days after setting. shellswithattachedjuvenilesintolargebottlescontaining
The subpopulation of oysters from the same cohort, 1G4F. After a minimum of 1 week, fixedjuveniles were
mm
which was reared separately to a mean size of 8.3 carefully pried from the large shell on which they had
shell length, was divided into groups ofbetween 15 and settled and were decalcified in formic acid. After decal-
25 and placed in molluscan rearing chambers designed cification and rinsing, animals were processed in paraffin
for conducting nutritional studies with young shellfish by standard methods (Humanson, 1979).
(Ukeles and Wikfors, 1982). Seawater supplying the P. minimum cultures were diluted with 1G4F fixative
chambers was filtered to 0.5 nm, passed through an ul- (10 ml/100 mlculture)andconcentratedbysettlinginan
traviolet sterilizer, and temperature-adjusted to 25 C in Imhoff cone. Supernatant was poured off, and concen-
a head tank. Seawater then was distributed to chambers trated cells were resuspended in 10 ml 1G4F for obser-
at a flow rate of600 ml min"1 through a flow-adjustable vation with transmission electron microscopy (TEM).
manifold. The same unialgal and mixed diets were fed to Thesecultureswereprocessed in paraffin with theagarose
juvenile oysters as were fed to larvae. Daily rations were embedding methods described above.
0.006 ml packed cells oyster"' cT', and percentage mixes Periodic acid-Schiff(PAS) staining ofplastic-embedded
were made by packed-cell volume, not cell number. A sectionswasaccomplished as follows: plastic was removed
full ration ofT-ISOconsistedof143 X 106cellsperoyster. from thick sections ofplastic-embedded larvae by incu-
316 G. H. WIKFORS AND R. M. SMOLOWITZ
TableI
Developmentofovsterembryosexposedfor48htocultured
phvloplanktonandcultureextracts(meansofthreereplicates. SDin
parentheses)
Added
OYSTERS EXPOSED TO A DINOFLAGELLATE 317
20
LARVAL COUNTS ALGAL DIETS
CD DEADABNORMAL (# = BAR IN CLUSTER)
ED DEAD NORMAL 1 - UNFED
15 EH LIVEABNORMAL 2 -T-ISO
LIVE NORMAL 3-1/3EXUV + 2/3T-ISO
4-2/3EXUV+ 1/3T-ISO
5-EXUV
Ill
1,0
T-ISO FED
OYSTERS SET
-
5
6 9 12 15 18 21 24 27 30
DAYS POST SPAWN
Figure 1. Countsofoysterlarvae(meansofthree replicates) from experimental feedingregimes.
Histologic observation of larvae from day 8 revealed ISO were in the prodissiconch II stage of development
clear differences between EXUV-fed, T-ISO-fed, mixed- (Elston, 1980) and demonstrated excellent cellularity of
diet, and unfed treatments (Figs. 3, 4). Animals fed on T- developing organs (Fig. 3A). Stomach and intestinal epi-
400
FEEDING REGIME: T-ISO FED
A OYSTERS SET
^__ T-ISO
2/3T-ISO + 1/3EXUV
300 _
1/3T-ISO + 2/3EXUV
rjj rj|
X
EXUV
_
CZD Q UNFED
200
LLJ
ILU
CO 100
^-9-9^ o
3 6 9 12 15 18 21 24 27 30 33
DAYS POST SPAWN
Figure 2. Shell-length measurementsoflarvae from experimental feedingregimes.
318 G. H. WIKFORS AND R. M. SMOLOWITZ
Table III Stomach and intestinal epithelia were cuboidal. Lipid
Growth rales, in micrometersperday(SE). oflan-aloysters droplets were rare in the stomach epithelium and very
Jedalga!diets rareintheintestinalepithelium. Digestiveglandsappeared
to be slightly more cellular than in animals fed EXUV
Algal diet Growth rate only, asdemonstrated by increased numbersofabsorptive
Unfed 0.744(,103)a cells within any section of the glands. Phagolysosomes,
EXUV as seen in control animals, and the distinctive accumu-
1.10 (.447)'
h EXUV 3.49 (.940)ab lation bodieswerenoted underbothlightandtransmission
EXUV
Vs 5.63 (1.33)^ electron microscopy.
T-ISO 6.52 (1.54)c
Superscnpt letters indicatehomogeneoussubsets, accordingto Least Newlyset spat
Square Difference Multiple RangeTest.
Only larvae fed T-ISO alone or '/3 EXUV + % T-ISO
developed into pedi-veligers and set, and only about 100
thelium was high-cuboidal in outline, and the intestinal spat were obtained with the latter diet including EXUV,
epithelium contained many prominent lipid droplets. The ascompared with morethan 2000, from threeinitial pop-
digestive gland contained numerous tall-columnar ab- ulations of2000, for the unialgal T-ISO diet. Spat from
sorptive cells. Examination with TEM showed that pha- thesetreatmentsweremaintained ontheirrespectivelarval
golysosomes within absorptive cell cytoplasm contained diets for 5 days after setting. Histologically, animals fed
moderately electron-dense flocculent substances(Fig. 4A). T-ISO alone exhibited well-developed organs with excel-
Rare phagolysosomes present at the basal side of some lent cellularity (Fig. 5A). The stomach epithelium was
cells contained dense, irregular inclusions. cuboidal, with rare, small lipidgranulesin thecytoplasm.
Starved animals were still in the prodissiconch I stage There were two to four profiles of intestine per section,
of development (Elston, 1980). All organ development and intestinal epithelial cells were cuboidal to columnar
was poor, and organs demonstrated low cellularity (Fig. in outline. Lipid droplets were abundant and prominent
3B). Stomach epithelium was low-cuboidal to squamous, in the intestinal epithelium. Phagocytes within the peri-
and intestinal profiles were rare. Lipid droplets were not visceral cavity contained clearto lightly foamy cytoplasm.
present in theseanimals. Thedigestiveglandsweresmall, Absorptive cells of the digestive gland were of the tall-
with poor cellularity, as demonstrated by the greatly re- columnar type, with foamy, vacuolated cytoplasm. Ex-
duced numbers of low-columnar to cuboidal absorptive amination with TEM demonstrated abundant phagoly-
cells. Examination with TEM showed that rare phagoly- sosomescontainingflocculent, irregular,granularmaterial
sosomes within the absorptive cell cytoplasm contained ofvarious densities, as noted in pre-set larvae fed T-ISO.
mildly flocculent contents. Newly set spat fed the mixed diet showed distinctive
All EXUV-fed larvae were in the late prodissiconch I and strikingdifferences from theT-ISOcontrols(Fig. 5A).
stageofdevelopment. Organ development andcellularity Animals contained moderately to poorly developed organ
was greater than in starved oysters, but was only about systems with reduced cellularity (Fig. 5B). Stomach epi-
one-third that ofT-ISO-fed animals (Fig. 3C). Stomach thelium was low-cuboidal to squamous and contained
and intestinal epithelium was cuboidal to sometimes moderate to heavy accumulations oflarge lipid droplets.
squamous. Lipid droplets were not seen in any cell type. Only one to two cross-sections ofintestine could be seen
Absorptive cells ofthe digestive glands were about one- in some sections. Intestinal cellswere cuboidal with vari-
third larger than in starved oysters. Interestingly, many able (moderate to small) amounts oflipid droplets in the
dense inclusions were present throughout the cytoplasm cytoplasm. Absorptive cells of the digestive gland were
ofthese cells (Fig. 3D). Examination with TEM showed cuboidal to low-columnar in outline. Both light and elec-
these inclusions to be a continuum of phagolysosome tron microscopic examination showed that the absorptive
profiles (which we have termed accumulation bodies) cells contained abundant accumulation bodies, as de-
from phagolysosomes containing possible autolysosome scribed above(Fig. 5C). Examination ofthe intestinal ep-
and otherdinotlagellate debris, to those filled with densely ithelium with TEM showed that it also contained mod-
flocculent material, to residual bodies containing hap- erate numbers ofaccumulation bodies.
hazardly arranged, laminated, dense bundlesoffiberswith Distinctive effects ofEXUV in the diet were seen in
both loose and condensed appearances (Fig. 4B and 4C). the perivisceral space of newly set oysters with both
Eight-day-old oysters fed a diet containing % EXUV light and transmission electron microscopy. Phagocytes
+ '/3 T-ISO were in the early prodissiconch II stage of within the space were plump and although some con-
development. Organ development and cellularity was tained only clear vacuoles. most phagocytes contained
slightly greater than in animals fed entirely on EXUV. various numbers oflarge, dense, irregular particles, 3-
OYSTERS EXPOSED TO A DINOFLAGELLATE 319
c
Figure3. Feedinglarvae. PhotomicrographsA, B,andCareallatthesamemagnification. Animalsare
embedded in plastic, thick-sectioned, and stained with methylene blue (1. digestive gland absorptivecells;
2,digestivegland enzymatic cell; 3. velum; 4, esophagus; 5. intestine; 6. style sac; 7, accumulation bodies
inabsorptivecells;8.lipidglobules;9.phagolysosomes).(A)Controlanimals(T-ISO-fed)showwell-developed
organs.(B)Starvedlarvaeshowpoordevelopmentofallorgans. (Cand D)Animalsfed 100% EXUVshow
poorly to moderately developed organswith distinctive accumulation bodies in the absorptive cells. (A to
C, bar = 20Mm; D, bar = 12.5 urn.)
10 nm in diameter. Other phagocytes surrounded and noflagellate. Examination with TEM showed partially
appeared to be in the process ofengulfing similar large, degraded dinoflagellates that contained small, round
irregular, dense particles (Fig. 6A). Rarely, less degen- bodies (possible autolysosomes) (Fig. 6B). Focal, mul-
erate particles showed profiles consistent with the di- ticellular necrosis within organs was not evident in
320 G. H. WIKFORS AND R. M. SMOLOWITZ
.4$rawifcswr"w-W'iV "iv"v^wTL*'-! J>' *,-''.--'. J^H
(SP|
fe; / r..-^'-'^ -^/^
Figure4. Feedinglarvae(transmissionelectron micrographs). (A)T-ISO-fed animalsshow moderately
electron-dense,flocculentsubstances!I)anddense,irregularinclusions(2)inphagolysosomes.(Bar=2^m.)
(BandC)Distinctiveaccumulationbodies(oysterphagolysosomescontainingdinoflagellateautolysosomes
and otherdebris) are present in absorptive cells ofEXUV-fed animals and show a progression from pha-
golysosomescontainingdebrisandroundbodiessimilartoautolysosomes(1),tophagolysosomescontaining
distinctiveflocculent material(2), to residualbodiescontainingdensebundlesoffibers(3).(B.bar = 2^m;
C,bar = 2fim.)
OYSTERS EXPOSED TO A DINOFLAGELLATE 321
5A
Figure5. Newlysetspat. Animalsareembedded inplastic,thick-sectioned,andstainedwith methylene
blue. (A) A T-ISO-fed control animal showsgood development oforgans with abundant lipid droplets in
the intestinal epithelium (I), columnar absorptive cells in the digestive glands (2). and rare phagocytes in
the perivisceral space (3). (Bar = 40^m.) (B and C) An EXUV-fed animal shows poor development of
intestineepithelium with vacuolation andlackoflipiddroplets(1). abundant lipiddropletsin thestyle-sac
epithelium (2). and distinctive accumulation bodies in the absorptive cells ofthe digestive gland (3). Di-
noflagellatesinvariousstagesofdegenerationarepresentwithinphagocytesorarebeingengulfedbyphagocytes
in thepenvisceral space(4). (B. bar = 40^m;C. bar = 11.4^m.)
EXUV-fed oysters; however, individual cell necrosis of tion bodies within the cytoplasm of absorptive cells of
the intestinal, digestive-gland, and stomach epithelium EXUV-fed (7A) but not T-ISO-fed oysters (Fig. 7B). The
appeared to be present. partially degraded dinoflagellates within the perivisceral
Histological examination of plastic-embedded larvae space ofnewly set spat fed the mixed diet were positively
stained with PAS showed positive staining ofaccumula- stained with PAS.
322 G H. WIKFORS AND R. M. SMOLOWITZ
B
Figure6. Newly setspat(transmissionelectron micrographs). (A)A perivisceral phagocyteofa mixed-
diet-fed animal shows engulfment and digestion ofthe dinoflagellates. (Bar = 5urn.) (B) A dinoflagellate
within the perivisceral spacehas numerousautolysosomes. (Bar = 1.25/im.)